Modulating the CDC14B-CDH1-PLK1 axis and methods for sensitizing target cells to apoptosis

ABSTRACT

The invention relates to modulating Cdc14B levels (cell division cycle 14 homolog B) and/or Cdh1 (Fzr1 protein, CDC20-like 1b, or fizzy-related protein) levels to sensitize cells to DNA damage by increasing the abundance of Plk1 (polo-like kinase 1) in a target cell. In certain embodiments, the invention relates to modulating Plk1 levels, and in particular to increasing Plk1 levels, to sensitize target cells such as cancer cells to cell death or apoptosis. In certain embodiments, the invention relates to inhibitors of Cdc14B and Cdh1 that sensitize tumor cells to chemotherapy or radiation induced cell death or apoptosis. In addition to applications relating to cancer therapies and diagnostics, the Plk1 modulators and assays will be employed for identifying novel drugs or drug candidates useful for various proliferative and/or differentiative disorders such as major opportunistic infections, immune disorders, cardiovascular diseases and inflammatory disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional. Patent Application Ser. No. 61/081,720, filed on Jul. 17, 2008, which is hereby incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part in the course of research sponsored by the National Institutes of Health (NIH) Grants R01-GM57587, R37-CA76584 and R21-CA125173. The U.S. government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to modulating Cdc14B levels (“cell division cycle 14 homolog B”) and/or Cdh1 levels (“fizzed related” “Fzr”) to sensitize cells to DNA damage by increasing the abundance of Plk1 (polo-like kinase 1) in a target cell. In certain embodiments, the invention relates to modulating Plk1 levels, and in particular to increasing Plk1 levels, to sensitize target cells such as cancer cells to cell death or apoptosis

BACKGROUND OF THE INVENTION The Ubiquitin Pathway

Ubiquitin-mediated proteolysis is an important pathway of non-lysosomal protein degradation which controls the timed destruction of many cellular regulatory proteins including, p27, p53, p300, cyclins, E2F, STAT-1, c-Myc, c-Jun, EGF receptor, IκBα, NFκB and β-catenin (reviewed in Pagano, 1997, FASEB J. 11: 1067). Ubiquitin is an highly conserved 76-amino acid polypeptide that is abundantly present in all eukaryotic cells. The ubiquitin pathway leads to the covalent attachment of a poly-ubiquitin chain to target substrates which are then degraded by the multi-catalytic proteasome complex (see Pagano, supra, for a recent review). Many of the steps regulating protein ubiquitination are known. Initially the ubiquitin activating enzyme (E1), forms a high energy thioester with ubiquitin which is, in turn, transferred to a reactive cysteine residue of one of many ubiquitin conjugating enzymes (Ubcs or E2s). The final transfer of ubiquitin to an e-amino group of a reactive lysine residue in the target protein occurs in a reaction that may or may not require an ubiquitin ligase (E3) protein. The large number of ubiquitin ligases ensures a high level of substrate specificity.

The Ubiquitin Pathway and the Regulation of the G1 Phase by F Box Proteins

Genetic and biochemical studies in several organisms have shown that the G1 phase of the cell cycle is regulated by the ubiquitin pathway. Proteolysis of cyclins, Ckis and other G1 regulatory proteins is controlled in yeast by the ubiquitin conjugating enzyme Ubc3 (also called Cdc34) and by an E3 ubiquitin ligase formed by three subunits: Cdc53, Skp1 and one of many F box proteins (reviewed in Patton, et al., 1998, Trends in Genet. 14:6). The F box proteins (FBPs) are so called because they contain a motif, the F Box, that was first identified in Cyclin F, and that is necessary for FBP interaction with Skp1 (Bai, et al., 1996, Cell 86:263). Cdc53 (also called Cul A) and Skp1 appear to participate in the formation of at least three distinct E3s, each containing a different FBP. Because these ligases are similar protein modules composed of Skp1, Cul A, and an FBP, they have been named SCF. The three SCFs identified in S. cerevisiae are: SCF^(Cdc4) (which recruits the Ckis Sic1 and Far1, the replication factor Cdc6, and the transcriptional activator Gcn4, as substrates through the F-Box protein Cdc4), SCF_(Grr1) (which recruits the G1 cyclins Cln1 and Cln2 as substrates through the F-Box protein GRR1), and SCF^(Met30) (which recruits the G1 cyclin Cln3 as a substrate throughout the F box protein MET30; see Pagano and Patton, supra, for recent reviews).

The interaction of SCF ligase with its substrates occurs via the FBP. FBPs are present in all eukaryotes (at least 54 in mammals; Cenciarelli, et al., 1999, Current Biol. 9: 1177; Winston, et al., 1999, Current Biol. 9: 1180). In addition to the F Box, many FBPs contain additional domains that facilitate both protein:protein interactions, e.g. WD-40 domains or leucine-rich repeats (LRRs), and protein:DNA interactions, e.g. tankyrase binding domains or HNH domains. Since the substrate specificity of SCF ligases is dictated by different FBPs that act as substrate targeting subunits, the large numbers of FBPs with varying combinations of protein or DNA interaction domains ensure highly specific substrate recognition.

FBP1, A Mammalian FBP Involved in Regulation of APC/C

Fbp1, the mammalian homolog of Xenopus β-TrCP1 (β-transducin repeat containing protein) (Spevak, et al., 1993, Mol. Cell. Biol. 8:4953), was identified using Skp1 as a bait in a two-hybrid screen (Cenciarelli, et al., supra). Fbp1 is an F box protein containing seven WD-40 domains (Margottin, et al., 1998, Mol. Cell 1:565), and is involved in the degradation of IκBα family members in response to NFκB activating stimuli (Gonen, et al., 1999, J. Biol. Chem. 274:14823; Hatakeyama, et al., 1999, Proc. Natl. Acad. Sci. USA 96:3859; Hattori, et al., 1999, J. Biol. Chem. 274:29641; Kroll, et al., 1999, J. Biol. Chem. 274:7941; Ohta, et al., 1999, Mol. Cell 3:535; Shirane, et al., 1999, J. Biol. Chem. 274:28169; Spencer, et al., 1999, Genes Dev. 13:284; Winston, et al., 1999, Genes Dev. 13:270; Wu and Ghosh, 1999, J. Biol. Chem. 274:29591; Yaron, et al., 1998, Nature 396:590). In addition, consistent with the finding that Xenopus and Drosophila Fbp1 orthologs act as negative regulators of the Wnt/β-catenin signaling pathway (Jiang and Struhl, 1998, Nature 391:493; Marikawa and Elinson, 1998, Mech. Dev. 77:75), several studies report that human Fbp1 controls β-catenin stability in vitro and in mammalian cultured cells (Hart, et al., 1999, Curr. Biol. 9:207; Hatakeyama, et al., supra; Kitagawa, et al., 1999, EMBO J. 18:2401; Latres, et al., 1999, Oncogene 18:849; Winston, et al., 1999, Genes Dev. 13:270).

Well-characterized substrates of mammalian Fbp1 have been found to share a common destruction motif, DSGxxS, and are recognized by Fbp1 only upon phosphorylation of the two serine residues present in this motif. There is, however, some recent evidence for additional mammalian substrates of Fbp1 lacking a completely conserved binding domain, such as ATF4 (Lassot, et al., 2001, Mol. Cell. Biol. 21:2192), Smad3 (Fukuchi, et al., 2001, Mol. Biol. Cell 12:1431), NFκB p105 (Orian, et al., 2000, EMBO J. 19:2580) and NFκB p100 (Fong and Sun, 2002, J. Biol. Chem. 277:22111). A conserved DSGxxS motif is present not only in Fbp1 substrates but also in certain regulators of Fbp1, such as the HIV protein Vpu, which targets Fbp1 to the non-physiological substrate, CD4, in virally infected cells. (Margottin, et al., supra). The DSGxxS destruction motif may also be found in peptide regulators of Fbp1 termed pseudosubstrates; however, pseudosubstrates escape the normal degradation fate of other FBP target proteins and instead modulate the activity of the FBP, and corresponding Cks, such as cellular localization and substrate targeting. For example, the Fbp1 pseudosubstrate hnRNP-U not only inhibits Fpb1 from targeting inappropriate substrates but also serves to localize Fbp1 to the nucleus (Davis, et al., 2002, Genes Dev. 16:439).

A further level of complexity is added by the presence of a Fbp1/β-TrCP1 (beta-transducin repeat containing protein 1) paralogous gene product, called β-TrCP2 (beta-transducin repeat containing protein 2) or Fbxw1B (78% identical, 86% similar to β-TrCP1; Kipreos and Pagano, 2000, Genome Biology 1:3002.1). Fbp1 and β-TrCP2 are ubiquitously expressed in adult human tissues (Cenciarelli, et al., supra; Koike, et al., 2000, Biochem. Biophys. Res. Commun. 269:103). In addition, β-TrCP2 has biochemical properties similar to Fbp1 in its ability to sustain the ubiquitinylation of both β-catenin and IKBα family members in vitro and to control their degradation in mammalian cultured cells (Fuchs, et al., 1999, Oncogene 18:2039; Suzuki, et al., 1999, Biochem. Biophys. Res. Commun. 256:127; Tan, et al., 1999, Mol. Cell 3:527). Despite these similarities, Fbp1 localizes to the nucleus and β-TrCP2 localizes mainly to the cytoplasm (Davis, et al., 2002, Genes Dev. 16:439). It is not clear whether these two FBPs have overlapping functions in vivo, or if each of them recognizes specific substrates.

Deregulation of the Ubiquitin Pathway in Cancer and Other Proliferative Disorders

Cancer develops when cells multiply too quickly. Cell proliferation is determined by the net balance of positive and negative signals. When positive signals overcome or when negative signals are absent, the cells multiply too quickly and cancer develops.

Ordinarily cells precisely control the amount of any given protein and eliminate the excess or any unwanted protein. To do so, the cell ubiquitinates the undesired protein to tag the protein for proteasome degradation. This mechanism goes awry in tumors, leading to the excessive accumulation of positive signals (oncogenic proteins), or resulting in the abnormal degradation of negative regulators (tumor suppressor proteins). Thus, without tumor suppressor proteins or in the presence of too much of an oncogenic protein, cells multiply without control, forming tumors (reviewed by Ciechanover, 1998, EMBO J. 17: 7151; Spataro, 1998, Br. J. Cancer 77: 448). For example, abnormal ubiquitin-mediated degradation of the p53 tumor suppressor (reviewed by Brown and Pagano, 1997, Biochim. Biophys. Acta 1332:1), the putative oncogene β-catenin (reviewed by Peifer, 1997, Science 275:1752) and the Cki p27 (reviewed in Ciechanover, supra; Spataro, supra; Lloyd, 1999, Am. J. Pathol. 154: 313) have been correlated with tumorigenesis, opening to the hypothesis that some genes encoding ubiquitinating enzymes may be mutated in tumors.

Initial evidence indicates that human F box proteins play a role in the ubiquitination of G1 regulatory proteins as do their homologues in yeast. Unchecked degradation of cell cycle regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligase plays a role in the altered degradation of cell cycle regulators. A well understood example is that of Mdm2, a ubiquitin ligase whose overexpression induces low levels of its substrate, the tumor suppressor p53.

Alternately, F box proteins have been shown to interact directly with DNA regulating proteins or DNA itself. F box proteins in yeast are known to regulate genomic stability and senescence, and recent data has shown that F box inhibition in mammalian cells can lead to the loss of DNA damage checkpoints.

DNA Damage and the Cell Cycle

The Anaphase Promoting Complex or Cyclosome (APC/C) is a ubiquitin ligase that plays a crucial role in the regulation of mitosis and the G1 phase of the cell cycle (Peters, 2006). In early mitosis, APC/C is activated through binding to Cdc20, and in late M, Cdc20 is replaced by Cdh1, the second activator of APC/C. During G1, APC/C^(Cdh1) remains active to ensure that certain positive regulators of the cell cycle do not accumulate prematurely. Then, at the G1/S transition, APC/C^(Cdh1) is inactivated by phosphorylation to allow stabilization of its substrates and promote progression into S phase. Cdk1-cyclin A and Cdk2-cyclin A mediate the phosphorylation of Cdh1, resulting in the dissociation of Cdh1 from the APC/C core (Lukas et al., 1999; Mitra et al., 2006; Sorensen et al., 2001). Other mechanisms inhibiting APC/C^(Cdh1) activity include Emi1 binding and degradation of both Cdh1 and Ubc10 (a ubiquitin conjugating enzyme that works with APC/C). During G2, both Cdh1 and Ubc10 reaccumulate, but APC/C^(Cdh1) remains inactive due to CDK-dependent phosphorylation of Cdh1 and the presence of Emi1. In early mitosis, Emi1 is eliminated via the SCF^(βTrCP) ubiquitin ligase, but the bulk of APC/C^(Cdh1) remains inactive due to high Cdk1 activity. Ultimately, Cdh1 activation in anaphase involves Cdk1 inactivation by APC/C^(Cdc20) and Cdh1 dephosphorylation. In yeast, this dephosphorylation is carried out by the Cdc14 phosphatase, but the mechanism in mammals remains unclear (D'Amours and Amon, 2004; Sullivan and Morgan, 2007).

Upon DNA damage, proliferating cells activate a regulatory signaling network to either arrest the cell cycle and enable DNA repair or, if the DNA damage is too extensive to be repaired, induce apoptosis (Bartek and Lukas, 2007; Harper and Elledge, 2007; Kastan and Bartek, 2004). The DNA damage response involves a number of factors that ultimately coordinate the spatiotemporal assembly of protein complexes at the site of DNA damage to initiate and maintain the checkpoint. Depending on the type of genotoxic stress, different checkpoint pathways are activated. UV and stalled replication forks activate the ATR-Chk1 pathway, whereas double-strand breaks result in the activation of both the ATM-Chk2 and the ATR-Chk1 pathways. After ATR is recruited to the site of damage, it phosphorylates and activates the effector kinase Chk1, a process requiring the mediator protein Claspin. Important downstream targets of Chk1 include p53 and Cdc25A, a transcription factor and an activator of Cdk1, respectively. Chk1-mediated phosphorylation induces the stabilization of p53 (with the consequent expression of the CDK inhibitor p21) and is required for the SCF^(βTrcp)-mediated degradation of Cdc25A; thus, Chk1 activation results in the attenuation of Cdk1 activity, with the consequent inhibition of mitosis.

During the recovery from DNA replication and DNA damage stresses, the G2 checkpoint must be silenced. This process involves the degradation of Claspin via the SCF^(βTrcp) ubiquitin ligase following the phosphorylation of Claspin by Plk1 (Mailand et al., 2006; Mamely et al., 2006; Peschiaroli et al., 2006). However, if DNA damage occurs during G2, SCF^(βTrcp)-dependent ubiquitylation of Claspin is inhibited to re-establish the checkpoint. The blocking of Claspin ubiquitylation is at least partially due to the inhibition of Plk1, which occurs in response to DNA damage (Smits et al., 2000). In fact, Claspin is not phosphorylated on its degron and does not bind βTrcp in G2 cells that have been subjected to DNA damage (Peschiaroli et al., 2006). However, despite the lack of SCF^(βTrcp)-Plk1-dependent ubiquitylation, Claspin continues to be ubiquitylated, only remaining stable due to a deubiquitylating enzyme (DUB), namely Usp28 (Zhang et al., 2006).

There is a general need for treatments relating to regulating or affecting the cell cycle to sensitize target cells, such as tumor or cancer cells, to cell death or apoptosis. In particular, there is a need for small molecule inhibitors that are useful as therapeutic agents, as well as for diagnostic and screening tools for identifying test therapeutic candidates.

SUMMARY OF THE INVENTION

The present invention provides a method of sensitizing a cell to cell death or apoptosis comprising contacting a target cell with an effective amount of an inhibitor of Cdc14B and/or Cdh1. In certain embodiments, the inhibitor results in an increase in the amount of protein compared to the amount of Plk1 protein prior to use of an effective amount of the inhibitor.

In certain embodiments, the invention relates to a method of sensitizing a cell to apoptosis or cell death comprising contacting the cell with an effective amount of an inhibitor of Cdc14B and/or Cdh1. In certain embodiments, the level of Plk1 protein (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) in the cell is increased compared to the amount of Plk1 in the cell prior to use of the inhibitor.

In certain embodiments, the cell is a diseased or abnormal cell from tissue or a cell line that exhibits a disease or abnormal condition selected from the group consisting of cancer, infection, immune disorder, cardiovascular disease, and inflammatory disorders.

In yet additional embodiments, the method further comprises contacting the cell with a second agent for sensitizing the cell to DNA damage, or for inducing apoptosis or cell death of a target cell.

In yet additional embodiments, the invention relates to a method of killing a cell comprising contacting the cell with an amount of an inhibitor of Cdc14B and/or Cdh1 that is effective to sensitize the cell to apoptosis or cell death.

In certain embodiments, the level of Plk1 protein (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) in the cell is increased compared to the amount of Plk1 in the cell prior to use of the inhibitor.

In yet additional embodiments, the method further comprises contacting the cell with a second agent for sensitizing the cell to DNA damage, or for inducing apoptosis or cell death of a target cell.

In further embodiments, the invention relates to a method of screening for an agent for sensitizing a target cell to apoptosis or cell death comprising: i) contacting a cell expressing Cdc14B and/or Cdh1 with a test compound; and ii) comparing the degradation rate of the Cdc14B and/or Cdh1 to a control, wherein the control is the degradation rate of Cdc14B and/or Cdh1 in the absence of the test compound; and iii) selecting a test compound that increases the degradation rate Cdc14B and/or Cdh1 as a compound for sensitizing a target cell to apoptosis or cell death.

In further embodiments, the invention relates to a method of screening for an agent for sensitizing a cancer cell to apoptosis or cell death: i) contacting a cell expressing Cdc14B and/or Cdh1 with a test compound; ii) comparing the amount of Cdc14B and/or Cdh1 present in the cell in the presence and in the absence of the test compound; and iii) selecting a test compound that decreases the amount of Cdc14B and/or Cdh1 in the cell as an agent for sensitizing a cancer cell to apoptosis or cell death.

In yet additional embodiments, the target cell is a diseased or abnormal cell from tissue or a cell line that exhibits a disease or an abnormal condition selected from the group consisting of cancer, infection, immune disorder, cardiovascular disease, and inflammatory disorders.

In further embodiments, the test compound is an siRNA. In certain embodiments, the siRNA comprises SEQ ID NO:20, SEQ ID NO:21, or a combination of both SEQ ID NO:20 and SEQ ID NO:21.

In yet additional embodiments, the Cdc14B or Cdh1 comprises the sequence of SEQ ID NO:15 or SEQ ID NO:10.

In further embodiments, the invention relates to a method of treating cancer in a mammalian subject which comprises: administering an effective amount of at least one Cdc14B or Cdh1 inhibitor to a mammalian subject suffering from cancer, wherein the inhibitor sensitizes cancer cells to chemotherapy.

In certain embodiments, the Cdc14B and/or Cdh1 inhibitor results in an increase in the amount of Plk1 protein (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) in a cell when compared to the amount of Plk1 protein in said cell prior to administering the inhibitor.

In yet additional embodiments, the invention relates to a kit for screening for an agent useful for modulating Plk1 protein (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) activity comprising: a Plk1 protein, at least one Plk1 binding protein selected from the group of Cdc14B (“cell division cycle 14 homolog B” also called “dual specificity protein phosphatase CDC14B”) and Cdh1 protein (“fizzed related”, “Fzr”), a means for detecting binding between the Plk1 and the Cdc14B and/or Cdh1 protein, and optionally instructions for use.

In further embodiments, the invention relates to a kit for screening for an agent useful for sensitizing a cell to apoptosis or cell death: a Plk1 protein (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) at least one Plk1 binding protein selected from the group of Cdc14B (“cell division cycle 14 homolog B” also called “dual specificity protein phosphatase CDC14B”) and Cdh1 protein (“fizzed related”, “Fzr”), a means for detecting binding between the Plk1 protein and the Cdc14B and/or Cdh1 protein, and optionally instructions for use.

In yet additional embodiments, the invention relates to a method for sensitizing a cell to apoptosis or cell death in a target cell of a mammal in need thereof, which comprises contacting said target cell with an effective amount of an inhibitor of Cdc14B and/or Cdh1.

In certain embodiments, the cell is a diseased or abnormal cell from a mammal that exhibits a disease or abnormal condition selected from the group consisting of cancer, infection, immune disorder, cardiovascular disease, and inflammatory disorders.

In yet additional embodiments, the method further comprises contacting the cell with a second agent for sensitizing the cell to DNA damage, or for inducing apoptosis or cell death of a target cell. In certain embodiments the mammal is a human.

In additional embodiments, the inhibitor of Cdc14B and/or Cdh1 comprises SEQ ID NO:20, SEQ ID NO:21, or a combination of both SEQ ID NO:20 and SEQ ID NO:21.

In yet further embodiments, the invention relates to a method of screening for an agent for sensitizing a target cell to apoptosis or cell death comprising: i) measuring the level of Cdc14B (“cell division cycle 14 homolog B” also called “dual specificity protein phosphatase CDC14B”) and/or Cdh1 levels (“fizzed related” “Fzr”) expressed by a target cell, ii) contacting the target cell that expresses Cdc14B and/or Cdh1 with a test compound, iii) measuring the amount of Cdc14B and/or Cdh1 expressed by the cell after said contacting step, iv) comparing the level of Cdc14B and/or Cdh1 expression in said cell before and after said contacting step, and v) selecting as a therapeutic agent candidate a test compound that decreases the level of Cdc14B and/or Cdh1 by said target cell.

In yet additional embodiments, the invention relates to a method of screening for an agent for sensitizing a target cell to apoptosis or cell death comprising: i) measuring the level of Plk1 (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) protein expressed by a target cell, ii) contacting the target cell that expresses Plk1 protein with a test compound, iii) measuring the amount of Plk1 protein expressed by the cell after said contacting step, iv) comparing the level of Plk1 expression in said cell before and after said contacting step, and v) selecting as a therapeutic agent candidate a test compound that increases the level of Plk1 protein by said target cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are immunoblots and corresponding cell cycle profiles showing SCF^(βTrcp)-independent degradation of Claspin in G1.

FIGS. 2A-D are immunoblots showing Claspin is degraded in G0 and G1 via the APC/C^(Cdh1) ubiquitin ligase.

FIGS. 3A-F show mapping of the Cdh1-binding motif in Claspin.

FIGS. 4A-B show that the N-terminus of Claspin is ubiquitylated in a Cdh1-dependent manner.

FIGS. 5A-I show reassociation and reactivation of APC/C^(Cdh1) upon exposure to genotoxic stress in G2.

FIGS. 6A-E are immunoblots and graphs showing that in response to DNA damage in G2, APC/C^(Cdh1) is reactivated to target Plk1 and Claspin, which is protected by Usp28.

FIGS. 7A-K show half-life analyses of Plk1 and Claspin under different conditions.

FIGS. 8A-H illustrate that DNA damage checkpoint in G2 requires Cdh1-dependent degradation of Plk1.

FIG. 9 shows expression of either a stable Claspin mutant or a stable Wee1 mutant prevents Plk1 from bypassing the checkpoint.

FIGS. 10A-F shows cells expressing stable Plk1 enter mitosis despite the presence of DNA damage.

FIGS. 11A-F illustrate that Cdh1 associates with Cdc14B in G2 in response to DNA damage.

FIGS. 12A-F illustrate that DNA damage-dependent reactivation of APC/C^(Cdh1) in G2 requires Cdc14B.

FIGS. 13A-I are immunoblots and graphs showing a constitutively active Cdh1 mutant is refractory to the silencing of Cdc14B.

FIG. 14 is a graph illustrating that co-silencing of Cdh1 and Cdc14B is not synergistic in bypassing the checkpoint.

FIGS. 15A-D are schematics and graphs showing that the Cdc14B-Plk1-Cdh1 axis controls the DNA damage response in G2 and is deregulated in human tumors.

FIGS. 16A-R show the analysis of mRNA levels of Cdc14B, Cdh1, and Plk1 in different human tumors.

DETAILED DESCRIPTION

In certain embodiments, the invention relates to modulating Cdc14B levels (“cell division cycle 14 homolog B” also called “dual specificity protein phosphatase CDC14B”) and/or Cdh1 levels (“fizzed related” “Fzr”) to sensitize cells to DNA damage by increasing the abundance of Plk1 (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) in a target cell. In certain embodiments, the invention relates to modulating Plk1 levels, and in particular to increasing Plk1 levels, to sensitize target cells such as cancer cells to cell death or apoptosis. In certain embodiments, the invention relates to inhibitors of Cdc14B and Cdh1 that sensitize tumor cells to chemotherapy or radiation induced cell death or apoptosis. In addition to applications relating to cancer therapies and diagnostics, the Plk1 modulators and assays can be employed for identifying novel drugs or drug candidates useful for various proliferative and/or differentiative disorders such as major opportunistic infections, immune disorders, cardiovascular diseases and inflammatory disorders.

In another aspect of the present invention, the phosphatase Cdc14B has been shown to translocate from the nucleolus to the nucleoplasm in response to genotoxic stress in G2 phase and induces the activation of the ubiquitin ligase APC/C^(Cdh1) (Anaphase Promoting Complex/Cyclosome and its activator Cdh1), with the consequent degradation of Plk1, a prominent promitotic kinase. This process induces the stabilization of Claspin and Wee1, because the proteolysis of these two proteins requires phosphorylation by Plk1. It was also shown that the stabilization of Claspin promotes the activation of Chk1, the stabilization of Wee1 attenuates Cdk1 activity. Consequently, inactivation of Cdc14B or Cdh1 increases the susceptibility of tumor cells to DNA damage. The elucidation of this mechanism of DNA damage response in G2 provides targets for killing tumor cells, and in particular cancer cells in cooperation with other DNA damaging or chemotherapeutic agents. In particular, these results show that modulating Plk1 activity, and in certain embodiments increasing the Plk1 activity provides a method for sensitizing cells to DNA damage leading to cell death or apoptosis of a target cell, including a cancer cell.

Identifying the Cdc14B-Cdh1-Plk1 axis as a mechanism by which Plk1 is inactivated in response to DNA damage in G2 provides a system for identifying agents and methods for potentiating the killing of cancerous cells. Certain aspects of the invention relate to using Cdc14B and Cdh1 to modulate the level of Plk1. In a particular embodiment, Cdc14B and/or Cdh1 are inactivated leading to the susceptibility of a tumor cell such as a cancer cell to DNA damage. In certain embodiments, the activity of Cdc14B and/or Cdh1 is blocked leading to the susceptibility of a tumor cell such as a cancer cell to DNA damage.

Certain embodiments of the present invention are not limited to any particular amino acid or nucleic acid sequence. In some embodiments, certain specified sequences are preferred.

Human β-TrCP1 has been sequenced and has an amino acid sequence according to Accession No. NP 378663 (SEQ ID NO:1); the β-TrCP1 coding sequence is Accession No. 033637 (SEQ ID NO:2), while a cDNA fragment corresponds to SEQ ID NO:3. The β-TrCP1 gene encodes a member of the F-box protein family which is characterized by an approximately 40 amino acid motif, the F-box (Fbox Motif β-TrCP1: DHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKK (SEQ ID NO:19). The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which function in phosphorylation-dependent ubiquitination. The F-box proteins are divided into 3 classes: Fbws containing WD-40 domains, Fbls containing leucine-rich repeats, and Fbxs containing either different protein-protein interaction modules or no recognizable motifs. The protein encoded by this gene belongs to the Fbws class; in addition to an F-box, this protein contains multiple WD-40 repeats. This protein is homologous to Xenopus β-TrCP1, yeast Met30, Neurospora Scon2 and Drosophila Slimb proteins. It interacts with HIV-1 Vpu and connects CD4 to the proteolytic machinery. It also associates specifically with phosphorylated IκBα and β-catenin destruction motifs, most likely functioning in multiple transcriptional programs by activating the NF-κB pathway and inhibiting the β-catenin pathway. Human β-TrCP1 is a variant that contains an additional 108 nt fragment within the coding region, as compared to human variant 2 (β-TrCP2), and thus encodes an in-frame 36 aa longer isoform than human variant β-TrCP2.

Human β-TrCP2 has been sequenced and has an amino acid sequence according to GenBank Accession No. 003930 (SEQ ID NO:4); the coding sequence is GenBank Accession No. 003939 (SEQ ID NO:5).

Human Cdc25A (cell division cycle 25A protein) has been sequenced and is a 524 amino acid long protein according to GenBank Accession No. NP_(—)001780 (SEQ ID NO:6); the coding sequence is GenBank Accession No. NM_(—)001789 (SEQ ID NO:7).

Human Claspin protein has been characterized and has a GenBank Accession No. NP_(—)071394.2 (SEQ ID NO:9). The coding sequence is found in GenBank Accession No. NM_(—)022111 (SEQ ID NO:8).

Human Cdh1 (also known as Fzr1 protein, CDC20-like 1b, or fizzy-related protein) has been characterized and has GenBank Accession No. NP_(—)057347 (SEQ ID NO:10). The coding sequence is found in GenBank Accession No. NM_(—)016263 (SEQ ID NO:11).

Human Cdc20 has been characterized as a protein that has GenBank Accession No. CAB92757 (UniProtKB/Swiss-Prot: Q12834 (SEQ ID NO:12). The coding sequence is contained within the genome locus Genbank Accession No. AL139289.

Human Cdc14B has been characterized and has at least three isoforms/variants. The isoform 1 protein has GenBank Accession No. NM_(—)003662 (SEQ ID NO:15). The coding sequence for isoform 1 corresponds to GenBank Accession No. NM_(—)003671. (SEQ ID NO:16). The isoform 2 protein has GenBank Accession No. NP_(—)201588 (SEQ ID NO: 30). The coding sequence for isoform 2 corresponds to GenBank Accession No. NM_(—)033331 (SEQ ID NO:31). The isoform 3 protein has GenBank Accession No. NP_(—)001070649 (SEQ ID NO:32). The coding sequence for isoform 3 corresponds to GenBank Accession No. NM_(—)001077181. (SEQ ID NO:33).

Human Cdc14A has been characterized as a protein that has GenBank Accession No. AAH38979 (SEQ ID NO:13). The coding sequence for Cdc14A corresponds to GenBank Accession No. BC038979 (SEQ ID NO:14).

Human Plk1 wildtype (wt) has been characterized as a protein and has GenBank Accession No. P53350. (SEQ ID NO:17). Mutants R337A and L340A are utilized in the present experiments. The coding sequence corresponds to GenBank Accession No. NM_(—)005030. (SEQ ID NO:18).

In accordance with the present invention there may be employed conventional molecular biology, microbiology, protein expression and purification, antibody, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. (2001) Molecular Cloning. A Laboratory Manual. 3^(rd) ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.; Nucleic Acid Hybridization, Hames & Higgins eds. (1985); Transcription And Translation, Hames & Higgins, eds. (1984); Animal Cell Culture Freshney, ed. (1986); Immobilized Cells And Enzymes, IRL Press (1986); Perbal, A Practical Guide To Molecular Cloning (1984); and Harlow and Lane. Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory Press: 1988).

DEFINITIONS

The following definitions are provided for clarity and illustrative purposes only, and are not intended to limit the scope of the invention.

The term apoptosis means a form of cell death in which a programmed sequence of events leads to the elimination of cells. Apoptosis plays an important role in developing and maintaining health by eliminating old cells, unnecessary cells, and unhealthy cells. The human body replaces perhaps a million cells a second. Too little or too much apoptosis plays a role in many diseases. When programmed cell death does not work properly, cells that should be eliminated may remain and become immortal. An example of the lack of proper apoptosis occurs for example, in cancer and leukemia. Impaired apoptosis is central for the development of cancer. Defects in apoptosis not only provide the cells an intrinsic survival advantage but also confer resistance to chemotherapeutic drugs. When apoptosis works too well, it kills too many cells and inflicts tissue damage. Apoptosis is also called programmed cell death or cell suicide.

As used herein, apoptosis, cell suicide, and programmed cell death are used interchangeably.

The term “cell death” is used generally to mean any type of cell death, and is not limited to programmed cell death or apoptosis

A “β-transducin repeat containing protein” or “β-TrCP” herein is a protein belonging to the family of F-box proteins containing 6-7 repeats of WD40 domains. Synonyms of β-TrCP1/2 include Fbw1a, FWD1a, Fbw1b, FWD1b, FBP1, and Hos. An F-box motif is a stretch of about 40 amino acids identified as being necessary for the interaction of F-box proteins with Skp1. The consensus sequence of an F-box motif is described in Bai et al., Cell, 1996; 86:263-274, hereby incorporated by reference in its entirety. A WD40 domain is a consensus sequence of about 40 amino acid repeats rich in tryptophan (Trp) and aspartic acid (Asp) residues (Neer et al., Nature, 1996; 371:297-300 and references therein, all of which hereby incorporated by reference in their entireties). A β-TrCP protein is characterized by being capable of a substrate specificity for at least one, preferably at least two, more preferably at least three, and most preferably at least all of phosphorylated Cdc25A, β-catenin, Emi1 (Guardavaccaro et al., Developmental Cell, 2003; 4:799-812), and IkB (Soldatenkov et al., Cancer Res., 1999; 59:5085-5088). A β-TrCP protein exhibits at least 50%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of the β-TrCP1 amino acid sequence (SEQ ID NO:1) or the β-TrCP2 amino acid sequence (SEQ ID NO:4), and includes functionally equivalent derivates of β-TrCP1 and β-TrCP2 such as mutants, conjugates (including radiolabeled or chemically tagged β-TrCP1/2), fusion proteins, and fragments thereof, which retain the substrate specificity of a β-TrCP. “β-TrCP1/2” means “β-TrCP1 and/or β-TrCP2”.

As used herein, a “β-TrCP inhibitor” is a compound or agent that causes one or more of the following: reducing β-TrCP1/2 expression, translation, or activity, or increasing β-TrCP1/2 degradation.

A “cell division cycle 25A” or “Cdc25A” protein herein means a protein comprising a peptide sequence corresponding at least to residues 82-88 of human wild-type Cdc25A (with reference to the full sequence, SEQ ID NO:6). Preferably, the peptide sequence comprises a sequence corresponding to residues 80-93 of human wild-type Cdc25A (with reference to the full sequence, SEQ ID NO:6). To function as a substrate for a β-TrCP, the serine residues corresponding to residues 82 and 88 of SEQ ID NO:6 must be at least phosphorylated, preferably double phosphorylated. Exemplary Cdc25A fragments useful for testing binding to or ubiquitination by β-TrCP1/2 include peptides corresponding to residues 73-95 of SEQ ID NO:6 and residues 80-93 of SEQ ID NO:6.

Cdc14B (“cell division cycle 14 homolog B” also called “dual specificity protein phosphatase CDC14B”). This protein is highly similar to Saccharomyces cerevisiae Cdc14, a protein tyrosine phosphatase involved in the exit of cell mitosis and initiation of DNA replication, which suggests the role in cell cycle control.

Cdh1 serves to sensitize cells to DNA damage by increasing the abundance of Plk1 (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) in a target cell. Cdh1 regulates ubiquitin ligase activity of the anaphase promoting complex/cyclosome (APC/C) confers substrate specificity upon the complex. The APC/C-Cdh1 dimeric complex is activated during anaphase and telophase and remains active in degrading substrates until onset of the next S phase.

Plk1 (polo-like kinase 1, also known as Plk, STPK13, or polo-like kinase homolog) is involved in the formation of and the changes in the mitotic spindle and in the activation of CDK/cyclin complexes during the M-phase of mitosis. Plk1 is involved in the formation of and the changes in the mitotic spindle and in the activation of CDK/cyclin complexes during the M-phase of mitosis.

A “DNA damaging agent” is a chemical compound or treatment method that induces DNA damage when applied to a cell, including single-strand breaks, double-strand breaks and alkylation. Such agents include, without limitation, ionizing radiation and waves that induce DNA damage, such as γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic agents”, function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Contemplated chemotherapeutic agents include alkylating agents such as mitomycin C, adozelesin, cis-platinum, and nitrogen mustard.

“Ubiquitin ligation”, “ubiquitination”, and “ubiquitinylation” as used herein all refer to the addition of a ubiquitin polypeptide to a protein substrate targeted for degradation.

About or Approximately

The term “about” or “approximately” means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term ‘about’ means within an acceptable error range for the particular value.

Administration

In the case of the present invention, parenteral routes of administration are also possible. Such routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, transmucosal, intranasal, rectal, vaginal, or transdermal routes. If desired, inactivated therapeutic formulations may be injected, e.g., intravascular, intratumor, subcutaneous, intraperitoneal, intramuscular, etc. In a preferred embodiment, the route of administration is oral. Although there are no physical limitations to delivery of the formulation, oral delivery is preferred because of its ease and convenience, and because oral formulations readily accommodate additional mixtures, such as milk and infant formula.

Adjuvant

As used herein, the term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Preferably, the adjuvant is pharmaceutically acceptable.

Amplification

“Amplification” of DNA as used herein denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al., Science 1988, 239:487.

Carrier

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

Coding Sequence or a Sequence Encoding an Expression Product

A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually, but not always, ATG) and a stop codon.

Dosage

The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. In some cases, oral administration will require a higher dose than if administered intravenously.

Expression Construct

By “expression construct” is meant a nucleic acid sequence comprising a target nucleic acid sequence or sequences whose expression is desired, operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) within the chosen host cells. Such sequence elements may include a promoter and a polyadenylation signal. The “expression construct” may further comprise “vector sequences.” By “vector sequences” is meant any of several nucleic acid sequences established in the art which have utility in the recombinant DNA technologies of the invention to facilitate the cloning and propagation of the expression constructs including (but not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.

Expression constructs of the present invention may comprise vector sequences that facilitate the cloning and propagation of the expression constructs. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic host cells. Standard vectors useful in the current invention are well known in the art and include (but are not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes. The vector sequences may contain a replication origin for propagation in E. coli; the SV40 origin of replication; an ampicillin, neomycin, or puromycin resistance gene for selection in host cells; and/or genes (e.g., dihydrofolate reductase gene) that amplify the dominant selectable marker plus the gene of interest.

Express and Expression

The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or secreted. The term “intracellular” means something that is inside a cell. The term “extracellular” means something that is outside a cell. A substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acid into a cell. The term “transformation” means the introduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cells genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

Expression System

The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

Gene or Structural Gene

The term “gene”, also called a “structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.

A coding sequence is “under the control of” or “operatively associated with” expression control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, particularly mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.

The term “expression control sequence” refers to a promoter and any enhancer or suppression elements that combine to regulate the transcription of a coding sequence. In a preferred embodiment, the element is an origin of replication.

Heterologous

The term “heterologous” refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. For example, the present invention includes chimeric DNA molecules that comprise a DNA sequence and a heterologous DNA sequence which is not part of the DNA sequence. A heterologous expression regulatory element is such an element that is operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed.

Homologous

The term “homologous” as used in the art commonly refers to the relationship between nucleic acid molecules or proteins that possess a “common evolutionary origin,” including nucleic acid molecules or proteins within superfamilies (e.g., the immunoglobulin superfamily) and nucleic acid molecules or proteins from different species (Reeck et al., Cell 1987; 50: 667). Such nucleic acid molecules or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of substantial percent similarity or the presence of specific residues or motifs at conserved positions.

Host Cell

The term “host cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays that are described infra. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal). Suitable host cells include but are not limited to Streptomyces species and E. Coli.

Immune Response

An “immune response” refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Such a response usually consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

Isolated

As used herein, the term “isolated” means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. Isolated nucleic acid molecules include, for example, a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic acid molecules also include, for example, sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. An isolated nucleic acid molecule is preferably excised from the genome in which it may be found, and more preferably is no longer joined to non-regulatory sequences, non-coding sequences, or to other genes located upstream or downstream of the nucleic acid molecule when found within the genome. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.

Mutant

As used herein, the terms “mutant” and “mutation” refer to any detectable change in genetic material (e.g., DNA) or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. As used herein, the term “mutating” refers to a process of creating a mutant or mutation.

Nucleic Acid Hybridization

The term “nucleic acid hybridization” refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are “hybridizable” to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). See Molecular Biology of the Cell, Alberts et al., 3^(rd) ed., New York and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length. Examples of high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., followed by washing in 0.1×SSC/0.1% SDS at 68° C. (where 1×SSC is 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide molecules washing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. (for 14 nucleotide-long oligos), at about 48° C. (for about 17 nucleotide-long oligos), at about 55° C. (for 20 nucleotide-long oligos), and at about 60° C. (for 23 nucleotide-long oligos)). Accordingly, the term “high stringency hybridization” refers to a combination of solvent and temperature where two strands will pair to form a “hybrid” helix only if their nucleotide sequences are almost perfectly complementary (see Molecular Biology of the Cell, Alberts et al., 3^(rd) ed., New York and London: Garland Publ., 1994, Ch. 7).

Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2×SSC at 65° C.; alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C.) and low stringency (such as, for example, an aqueous solution of 2×SSC at 55° C.), require correspondingly less overall complementarity for hybridization to occur between two sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).

As used herein, the term “standard hybridization conditions” refers to hybridization conditions that allow hybridization of sequences having at least 75% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

Nucleic acid molecules that “hybridize” to any desired nucleic acids of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, and at least 70 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as the particular desired nucleic acid.

Nucleic Acid Molecule

A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

Orthologs

As used herein, the term “orthologs” refers to genes in different species that apparently evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function through the course of evolution. Identification of orthologs can provide reliable prediction of gene function in newly sequenced genomes. Sequence comparison algorithms that can be used to identify orthologs include without limitation BLAST, FASTA, DNA Strider, and the GCG pileup program. Orthologs often have high sequence similarity. The present invention encompasses all orthologs of the desired protein.

Operatively Associated

By “operatively associated with” is meant that a target nucleic acid sequence and one or more expression control sequences (e.g., promoters) are physically linked so as to permit expression of the polypeptide encoded by the target nucleic acid sequence within a host cell.

Patient or Subject

“Patient” or “subject” refers to mammals and includes human and veterinary subjects.

Percent Sequence Similarity or Percent Sequence Identity

The terms “percent (%) sequence similarity”, “percent (%) sequence identity”, and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.), etc.

To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, Mass.; available at accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

In addition to the cDNA sequences encoding various desired proteins, the present invention further provides polynucleotide molecules comprising nucleotide sequences having certain percentage sequence identities to any of the aforementioned sequences. Such sequences preferably hybridize under conditions of moderate or high stringency as described above, and may include species orthologs.

Pharmaceutically Acceptable

When formulated in a pharmaceutical composition, a therapeutic compound such as an inhibitor of Cdc14B and/or Cdh1 can be admixed with a pharmaceutically acceptable carrier or excipient. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

Pharmaceutically Acceptable Derivative

The term “pharmaceutically acceptable derivative” as used herein means any pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, of a compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5^(th) Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. Preferred pharmaceutically acceptable derivatives are salts, solvates, esters, carbamates, and phosphate esters. Particularly preferred pharmaceutically acceptable derivatives are salts, solvates, and esters. Most preferred pharmaceutically acceptable derivatives are salts and esters.

Pharmaceutical Compositions and Administration

While it is possible to use a composition provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent and/or carrier. The excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine. The invention therefore includes within its scope pharmaceutical compositions comprising a product of the present invention that is adapted for use in human or veterinary medicine, including treating food allergies and related immune disorders.

In a preferred embodiment, the pharmaceutical composition is conveniently administered as an oral formulation. Oral dosage forms are well known in the art and include tablets, caplets, gelcaps, capsules, and medical foods. Tablets, for example, can be made by well-known compression techniques using wet, dry, or fluidized bed granulation methods.

Pharmaceutically acceptable excipients assist or make possible the formation of a dosage form for a bioactive material and include diluents, binding agents, lubricants, glidants, disintegrants, coloring agents, and other ingredients. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. An excipient is pharmaceutically acceptable if, in addition to performing its desired function, it is non-toxic, well tolerated upon ingestion, and does not interfere with absorption of bioactive materials.

Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The term “therapeutically effective amount” is used herein to mean an amount or dose sufficient to modulate, e.g., increase or decrease as appropriate, the expression or activity level of a desired protein e.g., by about 10 percent, preferably by about 50 percent, and more preferably by about 80-90 percent. In certain embodiments, the amount of Plk1 is increased as a result of decreasing the amount of Cdc14B and/or Cdh1. Preferably, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host following a therapeutic regimen involving one or more inhibitors of Cdc14B and/or Cdh1. The concentration or amount of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Suitable dosages may range from about 0.01 mg/kg to about 100 mg/kg of body weight per day, week, or month. The pharmaceutical compositions may also include other biologically active compounds.

According to the invention, a therapeutically effective amount of a Cdc14B and/or Cdh1 inhibitor can be formulated in a pharmaceutical composition of the invention to be introduced parenterally, transmucosally, e.g., orally, nasally, or rectally, or transdermally. Preferably, administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Optionally, the Cdc14B and/or Cdh1 inhibitor can be formulated together with an DNA damaging agent such as an alkylating agent.

In another embodiment, the active ingredient can be delivered in a vesicle, in particular a liposome (see Langer, Science, 1990; 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the therapeutic compound(s) can be delivered in a controlled release system. For example, a polypeptide may be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), a pellet containing a mixture of cholesterol and the active ingredient (Silastic®; Dow Corning, Midland, Mich.; see U.S. Pat. No. 5,554,601) implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.

The effective amounts of compounds of the present invention include doses that partially or completely achieve the desired therapeutic, prophylactic, and/or biological effect. The actual amount effective for a particular application depends on the condition being treated and the route of administration. The effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating and/or gastrointestinal concentrations that have been found to be effective in animals.

Kits

The invention provides a kit for screening for an agent useful for inhibiting Cdc14B and/or Cdh1 activity, comprising: a Plk1 protein, at least one of Cdc14B or Cdh1 protein, a means for detecting binding between the Plk1 and Cdc14B and/or Cdh1 protein, optionally packaged in association with instructions teaching one or more of the methods described herein. In certain embodiments, the invention provides a kit for screening for an agent useful for increasing the amount of Plk1 protein comprising: a Plk1 protein, at least one Cdc14B or Cdh1 protein, a means for detecting binding between the Plk1 and the Cdc14B and/or Cdh1 protein, optionally packaged in association with instructions teaching one or more of the methods described herein.

Polynucleotide or Nucleotide Sequence

A “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein). This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.

The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

Promoter

The promoter sequences may be endogenous or heterologous to the host cell to be modified, and may provide ubiquitous (i.e.+, expression occurs in the absence of an apparent external stimulus) or inducible (i.e., expression only occurs in presence of particular stimuli) expression. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839 and No. 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature 1981; 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 1980; 22:787-797), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 1981; 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 1982; 296:39-42); prokaryotic promoters such as the alkaline phosphatase promoter, the trp-lac promoter, the bacteriophage lambda P_(L) promoter, the T7 promoter, the beta-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 1978; 75:3727-3731), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA 1983; 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American 1980; 242:74-94; promoter elements from yeast or other fungi such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, and the PGK (phosphoglycerol kinase) promoter.

Small Molecule

The term “small molecule” refers to a compound that has a molecular weight of less than about 2000 Daltons, less than about 1000 Daltons, or less than about 500 Daltons. Small molecules, without limitation, may be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids, or other organic (carbon containing) or inorganic molecules and may be synthetic or naturally occurring or optionally derivatized. Such small molecules may be a therapeutically deliverable substance or may be further derivatized to facilitate delivery or targeting.

Substantially Homologous or Substantially Similar

In a specific embodiment, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 80%, and most preferably at least about 90% or 95% of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. An example of such a sequence is an allelic or species variant of the specific genes of the invention. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 80% of the amino acids are identical, or greater than about 90% are similar. Preferably, the amino acids are functionally identical. Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 10, Madison, Wis.) pileup program, or any of the programs described above (BLAST, FASTA, etc.).

Substantially Identical

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 80%, more preferably at least 90%, and most preferably at least 95% identity in comparison to a reference amino acid or nucleic acid sequence. For polypeptides, the length of sequence comparison will generally be at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably at least 50 amino acids. For nucleic acid molecules, the length of sequence comparison will generally be at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides.

The degree of sequence identity between any two nucleic acid molecules or two polypeptides may be determined by sequence comparison and alignment algorithms known in the art, including but not limited to BLAST, FASTA, DNA Strider, and the GCG Package (Madison, Wis.) pileup program (see, for example, Gribskov and Devereux Sequence Analysis Primer (Stockton Press: 1991) and references cited therein). The percent similarity between two nucleotide sequences may be determined, for example, using the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters.

Therapeutically Effective Amount

A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.

Therapeutically or Prophylactically Effective Amount of an Antibody

The compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antigen-binding portion of the desired inhibitor. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Transfection

By “transfection” is meant the process of introducing one or more of the expression constructs of the invention into a host cell by any of the methods well established in the art, including (but not limited to) microinjection, electroporation, liposome-mediated transfection, calcium phosphate-mediated transfection, or virus-mediated transfection.

Treating or Treatment

“Treating” or “treatment” of a state, disorder or condition includes:

-   -   (1) preventing or delaying the appearance of clinical or         sub-clinical symptoms of the state, disorder or condition         developing in a mammal that may be afflicted with or predisposed         to the state, disorder or condition but does not yet experience         or display clinical or subclinical symptoms of the state,         disorder or condition; or     -   (2) inhibiting the state, disorder or condition, i.e.,         arresting, reducing or delaying the development of the disease         or a relapse thereof (in case of maintenance treatment) or at         least one clinical or sub-clinical symptom thereof; or     -   (3) relieving the disease, i.e., causing regression of the         state, disorder or condition or at least one of its clinical or         sub-clinical symptoms.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

Variant

The term “variant” may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.

Vector, Cloning Vector and Expression Vector

The terms “vector”, “cloning vector” and “expression vector” refer to the vehicle by which DNA can be introduced into a host cell, resulting in expression of the introduced sequence. In one embodiment, vectors comprise a promoter and one or more control elements (e.g., enhancer elements) that are heterologous to the introduced DNA but are recognized and used by the host cell. In another embodiment, the sequence that is introduced into the vector retains its natural promoter that may be recognized and expressed by the host cell (Bormann et al., J. Bacteriol. 1996; 178:1216-1218).

Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A “cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Vector constructs may be produced using conventional molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The abbreviations in the specification correspond to units of measure, techniques, properties or compounds as follows: “min” means minutes, “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmole” means millimole(s), “kb” means kilobase, “bp” means base pair(s), “nt” means nucleotide, and “IU” means International Units. “Polymerase chain reaction” is abbreviated PCR; “Reverse transcriptase polymerase chain reaction” is abbreviated RT-PCR; “Estrogen receptor” is abbreviated ER; “DNA binding domain” is abbreviated DBD; “Untranslated region” is abbreviated UTR; “Sodium dodecyl sulfate” is abbreviated SDS; and “High Pressure Liquid Chromatography” is abbreviated HPLC.

Expression Plk1, Cdc14B and Cdh1 and Related Substrates

For the screening and evaluation of compounds for their ability to modulate the Plk1 interaction with Cdc14B and/or Cdh1, or other Plk1 substrates, both in vitro (including reconstituted systems) and in vivo systems (including cellular systems and transgenic animals) systems can be used. Regardless of the screening or testing system of choice, various expression methods can be employed to provide the protein components or cellular/transgenic animals to be used in the method.

A wide variety of host/expression vector combinations (i.e., expression systems) may be employed in expressing DNA sequences for Plk1, Cdc14B or fragments or mutants thereof, Cdh1 or fragments or mutants thereof, Cdc25A or fragments or mutants thereof. Additionally, DNA sequences expressing any of Skp1, Cul1, β-catenin, Emi1, IκB-α, IκB-β, IκB-ε, USP48, βTRCP1/2, Cdc 20, Cdc14B, or Fbxw 4-7, and other components are included as desirable host/expression vector combinations. These may be co-expressed from the same vector, expressed from different vectors, or one may be expressed while the other one is added externally to the screening or evaluation system. Useful expression vectors, for example, may consist of segments of chromosomal, non chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene, 1988; 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 m plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. In a preferred embodiment, various tumor cells lines can be used in expression systems of the invention.

Yeast expression systems can also be used according to the invention to express any protein of interest. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloning site; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), for example, can be employed according to the invention.

Expression of the protein or polypeptide may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839 and No. 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature, 1981; 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell, 1980; 22:787-797), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 1981; 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 1982; 296:39 42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 1978; 75:3727-3731), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A., 1983; 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980; 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogram et al., Nature, 1985; 315:338-340; Kollias et al., Cell, 1986; 46:89-94), hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al., Blood, 1991; 15:2557), etc.

Preferred vectors, particularly for cellular assays in vitro and in vivo, are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 1992; 7:980-990). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. Preferably, the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsidating the genome to produce viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci., 1991; 2:320-330), defective herpes virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No. WO 94/21807, published Sep. 29, 1994; International Patent Publication No. WO 92/05263, published Apr. 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 1992; 90:626-630; see also La Salle et al., Science, 1993; 259:988-990); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 1987; 61:3096-3101; Samulski et al., J. Virol., 1989; 63:3822-3828; Lebkowski et al., Mol. Cell. Biol., 1988; 8:3988-3996).

Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine 1995). In that regard, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem., 1992; 267:963-967; Wu and Wu, J. Biol. Chem., 1988; 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA, 1991; 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther.,1992; 3:147-154; Wu and Wu, J. Biol. Chem., 1987; 262:4429-4432). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. A relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has also been described (Mir et al., C.P. Acad. Sci., 1998; 321:893; WO 99/01157; WO 99/01158; WO 99/01175).

Another option is to transcribe and translate cDNA sequences in vitro. Various commercial systems are available for such techniques, including the TNT Quick Coupled Transcription/Translation System with Transcend™ (Promega, Madison, Wis.). For in vitro production of labeled or modified peptides or proteins, labeled or chemically modified amino acid precursors such as, e.g., ³⁵S-methionine or phosphoserine, can be added to the translation system.

Transgenic Animals

Transgenic mammals can be prepared for evaluating the interaction of human Plk1 with Cdc14B and/or Cdh1, or any other Plk1 substrates. Such mammals provide excellent models for screening or testing drug candidates, i.e., inhibitors of Cdc14B and/or Cdh1. Thus, human BimEL “knock-in” mammals can be prepared for evaluating the molecular biology of this system in greater detail than is possible with human subjects. In one embodiment, the animal can be double-transgenic, in that both human human Cdc14B and Cdh1 are expressed in the transgenic animal. It is also possible to evaluate compounds or diseases in “knock-out” animals, e.g., to identify a compound that can compensate for a defect in Cdc14B and/or Cdh1. Both technologies permit manipulation of single units of genetic information in their natural position in a cell genome and to examine the results of that manipulation in the background of a terminally differentiated organism. Trangenic mammals can be prepared by any method, including but not limited to modification of embryonic stem (ES) cells and heteronuclear injection into blast cells.

A “knock-in” mammal is a mammal in which an endogenous gene is substituted with a heterologous gene (Roemer et al., New Biol., 1991; 3:331). Preferably, the heterologous gene is “knocked-in” to a locus of interest, either the subject of evaluation (in which case the gene may be a reporter gene; see Elefanty et al., Proc. Natl. Acad. Sci USA, 1998; 95:11897) of expression or function of a homologous gene, thereby linking the heterologous gene expression to transcription from the appropriate promoter. This can be achieved by homologous recombination, transposon (Westphal and Leder, Curr. Biol., 1997; 7:530), using mutant recombination sites (Araki et al., Nucleic Acids Res, 1997; 25:868) or PCR (Zhang and Henderson, Biotechniques, 1998; 25:784).

A “knock-out mammal” is a mammal (e.g., mouse) that contains within its genome a specific gene that has been inactivated by the method of gene targeting (see, e.g., U.S. Pat. Nos. 5,777,195 and 5,616,491). A knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild type allele) and a homozygous mutant. Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo. A mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation. Zhou, et al. (Genes and Development, 1995; 9:2623 34) describes PPCA knock out mice. The term “knockout” refers to partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell. The term “knockout construct” refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. The nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous recombination (i.e., regions of the knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is incorporated into the corresponding position of the endogenous DNA). The knockout construct nucleic acid sequence may comprise (1) a full or partial sequence of one or more exons and/or introns of the gene to be suppressed, (2) a full or partial promoter sequence of the gene to be suppressed, or (3) combinations thereof. Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo. Generally, for homologous recombination, the DNA will be at least about 1 kilobase (kb) in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for recombination when the knockout construct is introduced into the genomic DNA of the ES cell.

Double knock-out mammals can be generated by repeating the procedures set forth herein for generating each knock-in or knock-out construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype. Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Lox system (see U.S. Pat. No. 4,959,317 and No. 5,801,030). The phrases “disruption of the gene” and “gene disruption” refer to insertion of a nucleic acid sequence into one region of the native DNA sequence (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell as compared to the wild type or naturally occurring sequence of the gene. By way of example, a nucleic acid construct can be prepared containing a DNA sequence encoding an antibiotic resistance gene which is inserted into the DNA sequence that is complementary to the DNA sequence (promoter and/or coding region) to be disrupted. When this nucleic acid construct is then transfected into a cell, the construct will integrate into the genomic DNA. Thus, many progeny of the cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the DNA is now disrupted by the antibiotic resistance gene.

In another series of embodiments, transgenic animals are created in which (i) a human Cdc14B and/or Cdh1 is stably inserted into the genome of the transgenic animal; and/or (ii) the corresponding endogenous genes are inactivated and replaced with their human counterparts (see, e.g., Coffman, Semin. Nephrol., 1997; 17:404; Esther et al., Lab. Invest., 1996; 74:953; Murakami et al., Blood Press. Suppl., 1996; 2:36). Such animals can be treated with candidate compounds and monitored for neuronal development, neurodegeneration, or efficacy of a candidate therapeutic compound.

Antibodies to Cdc14B and/or Cdh1

As described in the Examples, various antibodies useful for detecting Plk1, Cdc14B or Cdh1, or any of their substrates, have been produced, some of which are available commercially. Such antibodies may be used in immunoblotting or immunoprecipitation techniques to study binding of Cdc14B or Cdh1 to Plk1 or to another one of its substrates, to detect ubiquitinated Cdc25A, to inhibit interaction between Cdc14B and/or Cdh1 with Plk1, or one of its other substrates, or for other purposes in the screening and treatment methods described herein. Additional antibodies with different specificity or other particular properties may also be prepared. Antibodies useful for these purposes include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab expression library.

Various procedures known in the art may be used for the production of polyclonal antibodies. For example, various host animals can be immunized by injection with the antigenic polypeptide, including but not limited to rabbits, mice, rats, sheep, goats, etc. For preparation of monoclonal antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature, 1975; 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 1983; 4:72, Cote et al., Proc. Natl. Acad. Sci. U.S.A., 1983; 80:2026-2030), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690, published 28 Dec. 1989).

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to produce polypeptide-specific single chain antibodies. Indeed, these genes can be delivered for expression in vivo to, e.g., express an antibody inhibiting Plk1 interaction with Cdc14B and/or Cdh1. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science, 1989; 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a target polypeptide, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

Screening

A “test substance” or “test compound” is a chemically defined compound or mixture of compounds (as in the case of a natural extract or tissue culture supernatant), whose ability to modulate Plk1 activity or amount or that modulates the activity or the amount of Cdc14B and/or Cdh1 the a may be defined by various assays. A “test substance” is also referred to as a “candidate drug” or “candidate compound” in the present description.

Test substances may be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TIBTech, 1996; 14:60).

A modulatory effect may be determined by an in vitro method using a recombinant reporter gene promoter activity system including one or more of Plk1, Cdc14B, and Cdh1. Reporter genes for use in the invention encode detectable proteins, and include, but are by no means limited to, chloramphenicol transferase (CAT), β-galactosidase (β-gal), luciferase, green fluorescent protein (GFP) and derivatives thereof, yellow fluorescent protein and derivatives thereof, alkaline phosphatase, other enzymes that can be adapted to produce a detectable product, and other gene products that can be detected, e.g., immunologically (by immunoassay).

A screen according to the invention involves detecting expression of the reporter gene by the host cell when contacted with a test substance. If there is no change in expression of the reporter gene, the test substance is not an effective modulator. If reporter gene expression is modified, the test substance has modulated, e.g., increased Plk1-mediated gene expression, or decreased Cdc14B and/or Cdh1 expression, the test substance is a candidate for development as an agent capable of inducing apoptosis or cell death. Likewise, any such modulator is a candidate for use as a tumor sensitizing agent. Additionally, any such modulator is a candidate for use in combination with another DNA damaging or chemotherapeutic agent to facilitate tumor sensitization leading to cell death. The reporter gene assay system described herein may be used in a high-throughput primary screen for antagonists, or it may be used as a secondary functional screen for candidate compounds identified by a different primary screen, e.g., a binding assay screen that identifies compounds that decrease Cdc14B and/or Cdh1 transcription activity, and/or increase Plk1 activity.

Potential drugs may be identified by screening in high-throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time (see, e.g., U.S. Pat. Nos. 5,585,277, 5,679,582, and 6,020,141). Such high-throughput screening methods are particularly preferred. Alternatively, simple reporter-gene based cell assays such as the one described here are also highly desirable.

Intact cells or whole animals expressing genes encoding at least one of Plk1, Cdc14B and/or Cdh1, and optionally also any of the remaining components of an SCF complex, can be used in screening methods to identify candidate drugs. In one series of embodiments, a permanent cell line is established. Alternatively, cells are transiently programmed to express a Plk1 gene by introduction of appropriate DNA or mRNA. As described herein, transgenic animals can also be used to screen for or study agents that increase the level of Plk1. Similarly, it may be desirable for cells to be transiently programmed to express any of the genes encoding Cdc14B and/or Cdh1 by introduction of appropriate DNA or mRNA. As described herein, transgenic animals can also be used to screen for or study agents that increase the level of Cdc14B and/or Cdh1.

Identification of candidate substances can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to Plk1, to the Plk1 binding site on Cdc14B and/or Cdh1, or another one of its substrates (ii) assays that measure the ability of a test substance to modify (e.g., inhibit) a measurable activity or function of Cdc14B and/or Cdh1, (iii) assays that measure the ability of a substance to modify (i.e., inhibit) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions of at least one of the genes encoding Cdc14B and/or Cdh1; and (iv) assays that modulate (e.g., promote) the degradation of at least Cdc14B and/or Cdh1 proteins, while increasing the level of Plk1.

Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways, e.g. to enhance their proteolytic stability.

RNA Interference (RNAi or siRNA)

Another technique of interest for therapeutic purposes is based on the same principles employed for interfering with Cdc14B and/or Cdh1 translation in a cellular system, namely siRNA technology. Particularly, expression of selected genes can be suppressed in human cells by transfecting with exogenous, short RNA duplexes (siRNA) where one strand corresponds to a target region of the mRNA, i.e., EST of interest (Elbashir et al., Nature, 2001; 411:494-498). The siRNA molecules are typically greater than 19 duplex nucleotides, and upon entry into the cell, siRNA causes the degradation of single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. siRNA is more potent than standard anti-sense technology since it acts through a catalytic mechanism. Effective strategies to deliver siRNAs to target cells in cell culture include physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors (Scherr et al., Curr. Med. Chem., 2003; 10(3):245-56). Recently, the RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (Abbas-Turki et al., Hum. Gene Ther., 2002; 13(18):2197-201). siRNA can also be delivered in a viral vector derived, e.g., from a lentivirus (Tiscornia et al., Proc. Natl. Acad. Sci. U.S.A., 2003; 100:1844-8). For review articles, see Hannon, Nature, 2002; 418:244-51 and Bernstein et al., RNA, 2001; 7(11):1509-21. This technology also has been described in vitro in cultured mammalian neurons in Krickevsky and Kosik, Proc. Natl. Acad. Sci. USA, 2002; 99(18):11926-9. siRNA technology is also being used to make transgenic animals (Cornell et al., Nat. Struct. Biol., 2003; 10(2):91-2). RNAi is described in Publication Nos. WO 99/49029 and WO 01/70949.

Exemplary siRNA's suitable for β-TrCP1/2, Plk1, Cdc14B, or Cdh1 include:

(SEQ ID NO: 19) for human β-TrCP1/2: GUGGAAUUUGUGGAACAUC (SEQ ID NO: 20) for mouse β-TrCP1/2: AUCAAGAUCAGGGAUAAAA for human Cdc14B (SEQ ID NO: 22) for human Cdh1: (SEQ ID NO: 21). Materials & Methods

The following describes the materials and methods employed in Examples 1-6.

Cells

HeLa (human carcinoma; obtained from ATCC) or U2OS (human osteosarcoma) cells are used in certain of the Examples. Cell culture is conducted essentially as described in Donzelli et al. (Embo J, 2002; 21:4875-84). Cells are grown at 37° C. in a 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% bovine calf serum (Hyclone) and 2 mM L-glutamine (Euroclone), or in DMEM containing 5% FCS.

Cell Synchronization and Drug Treatments

U2OS and T98G cells were synchronized as described (Dorrello et al., 2006; Peschiaroli et al., 2006). Pulse treatment of U2OS cells with doxorubicin was performed for one hour at a final concentration of 0.5 μM. To measure protein half-lives, G2 cells were incubated in the presence of 100 μg/ml cycloheximide dissolved in 100% ethanol.

Cell Culture

U2OS, HeLa, HeLa-S3, T98G, and HEK293T cells were cultured as described (Dorrello et al., 2006; Guardavaccaro et al., 2003). ATM+/+ (NHF1-hTERT) and ATM−/− (GM0252A-hTERT) fibroblasts were grown as described by (Heffernan et al., 2002).

Antibodies

Mouse monoclonal antibodies were from Zymed/Invitrogen (anti-Cul1, anti-Plk1), Sigma (anti-FLAG, anti-Cdc27, anti-Cdh1), Santa Cruz Biotechnology (anti-Chk1, anti-Geminin, anti-Cdc25A), Boston Biochem (anti-UbcH10), BD Biosciences (anti-p27), Covance (anti-HA), and Abcam (anti-GFP). The mouse monoclonal antibodies against Claspin and USP28 were kind gifts from Thannos Halazonetis and Steve Elledge. Rabbit polyclonal antibodies were from Zymed/Invitrogen (anti-Cks1), Upstate (anti-phospho-Ser10 Histone H3, anti-phospho-Ser 139 Histone H2AX), Santa Cruz Biotechnology (anti-Wee1, Cdk2, phospho-Tyr15 Cdk1, Skp1, Bub1), and Cell Signaling (phospho-Ser317 Chk1, phospho-Thr68 Chk2). The rabbit polyclonal antibodies against TMPK and TK1 were a kind gift from Zee-Fen Chang. Rabbit polyclonal antibodies against Cdk1 (Carrano et al., 1999), cyclin A (Carrano and Pagano, 2001), and the mouse monoclonal antibody to cyclin E (Faha et al., 1993) were previously described.

In Vitro Ubiquitylation Assay

Ubiquitylation assays were previously described (Bashir et al., 2004). Briefly, an anti-Cdc27 antibody was added to cell extracts and incubated for approximately 3 hours at 4° C. Protein G-agarose was then added and incubated for 45 minutes at 4° C. on a rotating wheel. The beads were washed 4 times in Triton buffer and 4 times in QA buffer (10 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM MgCl₂, 0.1 mM CaCl₂, 1 mM DTT). The resulting beads were used for two reactions of in vitro ubiquitylation. Ubiquitylation assays were performed in a volume of 10 μl containing 50 mM Tris pH 7.6, 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 2 μl in vitro transcribed/translated unlabeled Cdh1, 50 ng/μl E1 (Boston Biochem), 100 ng/μl Ubc, 100 ng/μl Ubc10, 2.5 μg/μl ubiquitin (Sigma), 1 μM ubiquitin aldehyde, and 1 μl ³⁵S-methionine-labelled in vitro transcribed/translated substrate [i.e. wild type NT-Claspin, NT-Claspin(ENL), CT-Claspin, Plk1, or cyclin B labeled in vitro transcribed/translated in rabbit reticulocyte lysate (RRL)]. The reactions were incubated at 30° C. for the indicated times and analyzed by SDS-PAGE and autoradiography. The RRLs containing the in vitro transcribed/translated substrates were treated with 5 mM NEM prior to ubiquitylation reactions, which transitorily inactivates ubiquitylating activities present in the RRL (Rodrigo-Brenni and Morgan, 2007). The assays shown in Figure S4D did not contain Cdh1.

GST Fusion Proteins and Pull-Down Assay

GST-Cdh1 or GST-Claspin mutants were expressed in E. coli (BL-21) using the pGEX4T2 vector (Amersham). For protein purification, bacteria were grown to an optical density of 600 nm in Luria-Bertani medium, induced at 37° C. with 0.1 mM isopropyl-1-thio-D-galactopyranoside, and cultivated for 2 h. Bacteria were then pelleted, resuspended in NETN-buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris.HCl [pH 7.4], 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine), and sonicated. Insoluble material was removed by centrifugation. Thirty microliters of Glutathione-S-Sepharose 4b beads (Amersham) were added to the cleared lysate, incubated for 30 min at 4° C., and washed 3 times with NETN-buffer. GST pull-down assays were performed as described previously (Bassermann et al., 2005). Briefly, wild type and mutant Claspin proteins or Cdh1 were in vitro transcribed/translated and ³⁵S-radiolabeled using the TNT system (Promega). GST fusion proteins were added and incubated for one hour at 4° C. Subsequently, protein complexes were washed thoroughly with NETN buffer, subjected to SDS-PAGE, and visualized by autoradiography.

Plasmids

cDNAs of wild type Claspin, Claspin point- and deletion-mutants, βTrcp1/2, Fbxw4, Fbxw5, Fbxw6, Fbxw7, Cdh1, and Cdc20 were cloned into pcDNA 3.1. pGFP-Cdc14A and pGFP-Cdc14B were kind gifts from Jiri Lukas. For retrovirus production, Cdc14B, Cdh1 WT, and Cdh1(4xA) were subcloned into the retroviral vector pMSCV while Plk1 WT and Plk1(R337A, L340A) were subcloned into the retroviral vector pBabe. All cDNAs were sequenced. Point mutants were generated using the QuikChange Site-directed Mutagenesis kit (Stratagene), and deletion mutants were prepared by standard PCR procedures.

siRNA Oligos

The sequences of the oligonucleotides corresponding to βTrcp1/βTrcp2, Cdh1, Cdc14B, and Usp28 mRNAs were GUGGAAUUUGUGGAACAUC (SEQ ID NO:20), UGAGAAGUCUCCCAGUCAG (SEQ ID NO:21), GAUGCUACAUGGUUAUAUA (SEQ ID NO: 22), and CUGCAUUCACCUUAUCAUU (SEQ ID NO:23), respectively. These dsRNA oligos have been previously validated: Cdh1 (Bashir et al., 2004; Brummelkamp et al., 2002; Donzelli et al., 2002; Ke and Chang, 2004), Usp28 (Zhang et al., 2006), βTrCP1/2 (Dorrello et al., 2006; Fong and Sun, 2002; Guardavaccaro, 2008; Guardavaccaro et al., 2003; Jin et al., 2003; Peschiaroli et al., 2006) and Cdc14B (Rodier et al., 2008). A dsRNA oligo to LacZ mRNA (CGUACGCGGAAUACUUCGA) (SEQ ID NO: 24) served as control.

mRNA Analysis

RNA was extracted using the RNeasy Kit (Qiagen). cDNA synthesis was performed using Superscript III (Invitrogen). Quantitative PCR analysis was performed according to standard procedures. Primer sequences were:

5′GTGCCATTGCAGTACATT3′ (SEQ ID NO: 25) and 5′AGCAGGCTATCAGAGTG3′ (SEQ ID NO: 26) (Cdc14B) and 5′CGCCGCTAGAGGTGAAATTC3′ (SEQ ID NO: 27) and 5′CTTTCGCTCTGGTCCGTCTT3′ (SEQ ID NO: 28) (18S rRNA). Immunofluorescence Microscopy

Direct and indirect immunofluorescence was performed as described (Frescas et al., 2007). Primary antibodies (anti-FLAG, Sigma; anti-GFP, Abcam) were used at a dilution of 1:1000.

In Vivo Labeling with Orthophosphate

U2OS cells expressing either wild type Cdh1 or Cdh1(4xA) were transfected with control or Cdc14B siRNA oligonucleotides and synchronized at G1/S using a double thymidine block. Seven hours post release, cells were washed twice in labeling medium [(phosphate-free DMEM, supplemented with 10% dialyzed serum (Hyclone)] and subsequently incubated for three hours in labeling medium, containing 0.5 μM doxorubicine and [³²P]-orthophosphate (0.5 mCi/ml, Perkin Elmer). Denatured cell extracts were subsequently prepared in 1% SDS. Prior to immunoprecipitation with anti-FLAG agarose (Sigma), cell extracts were diluted 10-fold with lysis buffer containing 1% Triton X-100.

Data Mining

Gene expression data on Cdc14B, Cdh1 and Plk1 were retrieved from the Oncomine website. Data was re-analyzed in GraphPad software to show expression levels of Cdc14B, Cdh1, and Plk1 for each cancer study. GraphPad software was used to determine P values. Additional details relating to these studies, including the pathological and clinical data, are available at Oncomine or via the individual journal websites. Kaplan-Meier survival curves of 219 brain cancer patients were obtained from the Repository of Molecular Brain Neoplasia Database Rembrandt website of the National Cancer Institute at NIH. Kaplan-Meier survival plots were generated by grouping gliomas of all histological grades by the gene expression levels of Cdc14B, Cdh1 and Plk1. Additional details relating to these studies, including the pathological and clinical data of individual patients, are available on the Rembrandt website.

Normalization and Quantification of Protein Levels

Protein concentrations of whole cell extracts (WCE) were performed using a Bio-Rad DC protein assay (Lowry assay) according to the manufacturers instructions. For each experiment, equal amounts of WCE (in general, 30 μg) were separated by SDS-PAGE and then analyzed by immunoblotting. Equal protein levels in each lane were confirmed by Ponceau S staining of the membrane and by immunoblotting a protein whose levels are not regulated by either DNA damage or during cell cycle progression (e.g., Cul1 or Skp1). To make the assay as linear as possible, densitometric quantification of bands was performed using Quantity One software (Bio-Rad) on low-saturation exposures. To be able to directly compare protein levels of different gels and independent experiments, an equal WCE (e.g., 30 μg of HeLa WCE) was loaded in each gel as a standard reference.

EXAMPLES

The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Claspin is a Substrate of APC/C^(Cdh1) in the G1 Phase of the Cell Cycle.

It was previously known that the levels of Claspin oscillate throughout the cell cycle (Mailand et al., 2006; Mamely et al., 2006; Peschiaroli et al., 2006). The highest Claspin expression levels are observed in S phase and early G2, and levels decrease thereafter, becoming almost undetectable during mitosis and the following G1 phase, as shown in FIGS. 1A-E. In FIG. 1A U2OS cells were released from a prometaphase arrest (indicated as time 0) and collected at the indicated times. Protein extracts were analyzed by immunoblotting with antibodies to the indicated proteins. (AS, asynchronous cells). FIG. 1B shows cell cycle profiles of the cells used in the experiment shown in FIG. 1A as determined by flow cytometry.

For the experiments shown in FIG. 1C, T98G cells were switched to culture media containing 0.02% FBS to arrest them in G0/G1. Samples were collected at the indicated times after the beginning of the serum starvation and subjected to immunoblot analysis using antibodies to the indicated proteins. FIG. 1D shows cell cycle profiles of the cells used in the experiment shown in FIG. 1C as determined by flow cytometry.

In FIG. 1E U2OS cells were transfected with siRNA oligos to both βTrcp1 and βTrcp2 mRNAs. Cells were collected after being synchronized in prometaphase (PM) or G1 phase, and cell extracts were analyzed by immunoblotting with antibodies to the indicated proteins.

Levels of Claspin also decrease when cells withdraw from the cell cycle and enter quiescence (FIG. 1B). Since SCF^(βTrcp) mediates the degradation of Claspin degradation at G2/M, experiments were designed to determine whether this ligase is also responsible for Claspin degradation in G1. Both βTrcp1 and βTrcp2 were downregulated using an established siRNA oligo and Claspin levels in both M and G1 were analyzed.

While silencing of βTrcp induced an accumulation of Claspin in prometaphase cells, no effect was visible in G1 cells (FIG. 1E), suggesting that a different ligase from SCF^(βTrcp) targets Claspin for degradation in G0 and G1. Thus, experiments were designed to identify the uncharacterized G1 specific ubiquitin ligase by investigating the ability of Claspin to bind ubiquitin ligase subunits. The data showed that only Cdh1 was able to co-immunoprecipitate endogenous Claspin, whereas Fbxw4, Fbxw5, Fbxw6, Fbxw7, and Cdc20 (FIG. 2A), as well as thirteen different F-box proteins (Peschiaroli et al., 2006), failed to bind Claspin.

To test whether APC/C^(Cdh1) has a role in the degradation of Claspin during G0/G1, the expression of Cdh1 was reduced in T98G cells using a validated isRNA (SEQ ID NO:21). T98G cells were serum starved following knockdown of Cdh1, and the expression levels of Claspin were analyzed at various time points thereafter. FIGS. 2A-D are immunoblots showing Claspin is degraded in G0 and G1 via the APC/C^(Cdh1) ubiquitin ligase. FIG. 2A shows that Cdh1 interacts with Claspin in vivo. HEK293T cells were transfected with the indicated FLAG-tagged constructs or an empty vector (EV). Whole cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with antibodies to the indicated proteins.

FIG. 2B shows T98G cells were transfected with a control (Ctrl) siRNA oligo or an siRNA oligo directed against Cdh1 mRNA. Cells were then switched to culture media containing 0.02% FBS to arrest them in G0/G1. Samples were collected at the indicated times after the beginning of the serum starvation and subjected to immunoblot analysis using antibodies to the indicated proteins. Skp2 and p27 were used as cell cycle markers.

FIG. 2C shows Claspin(ENL) is stable in G1. HeLa cells were infected with retroviruses expressing either FLAG-tagged wild type Claspin or FLAG-tagged Claspin(ENL) and subsequently treated for 16 hours with nocodazole to induce a mitotic block. Round, prometaphase cells were then collected by gentle shake-off and replated in fresh medium for the indicated times. Cells were harvested, and cell extracts were analyzed by immunoblotting with antibodies to the indicated proteins. Synchronization was monitored by flow cytometry and by the levels of cyclin B.

FIG. 2D shows Ubiquitin ligation assays of ³⁵S-labeled, in vitro translated Claspin N-terminal fragments (amino acid 1-678) of either wild type Claspin or Claspin(ENL) were conducted in the presence of unlabeled, in vitro translated Cdh1. Samples were incubated at 30° C. for the indicated times. The lower autoradiography image represents a short exposure time, and the upper image represents a long exposure time. The bracket on the right side of the top panel marks a ladder of bands corresponding to polyubiquitylated Claspin.

These experiments illustrated that downregulation of Cdh1 strongly inhibited the degradation of Claspin in cells progressively accumulating in G0/G1 (FIG. 2B). Similarly, T98G cells released from a block in prometaphase retained Claspin expression throughout G1 phase upon silencing of Cdh1. Together, these data show a role for APC/C^(Cdh1) in targeting Claspin for degradation during G0 and G1.

The Cdh1 binding motif of Claspin was systematically mapped. In FIG. 3A the indicated deletion mutants were transcribed/translated in vitro and tested for their binding to GST-tagged Cdh1 using in vitro pull-down assays. Claspin mutants that were pulled-down by GST-Cdh1 were separated by SDS-PAGE and visualized by autoradiography and designated with the symbol (+).

In FIG. 3B HEK293T cells were co-transfected with HA-tagged Cdh1 and the indicated FLAG-tagged deletion mutants of Claspin. Whole cell extracts (WCE, bottom panel) were immunoprecipitated (IP, upper two panels) with an anti-HA antibody, and the indicated proteins were detected by immunoblotting. FIG. 3C shows a schematic representation of the in vivo binding data shown in FIG. 3B.

In FIG. 3D in vitro transcribed/translated, ³⁵S-labeled Cdh1 was assayed for in vitro binding to the indicated GST-tagged Claspin fragments. Bound Cdh1 was separated by SDS-PAGE and visualized by autoradiography. This series of binding experiments using multiple Claspin deletion mutants narrowed the binding motif to an N-terminal region located between amino acids 79-102 of SEQ ID NO:9 (FIGS. 3A-D).

FIG. 3E shows CLUSTALW alignment of Claspin orthologs with shading of conserved amino acids. Dark gray: identical residues; light gray: similar residues. Five groups of three amino acids mutated individually in Claspin are framed.

HEK293T cells were cotransfected with HA-tagged Cdh1 and the indicated FLAG-tagged Claspin mutants. Whole cell extracts (WCE, bottom two panels, FIG. 3B) were immunoprecipitated (IP, upper two panels FIG. 3B) with an anti-HA antibody, and the indicated proteins were detected by immunoblotting. Finally, five unique, triple-point-mutations to Alanine across Claspin amino acids 79-102 were inserted, following the pattern of evolutionary conserved residues (FIG. 3E), and these mutants were assayed for binding to Cdh1. The immunoblots are shown in FIG. 3F. Both mutant #2 [Claspin(EEN)] and mutant #3 [Claspin(ENL)] failed to bind Cdh1 (FIG. 3F). Thus, these studies identified the motif “EENxENL” (SEQ ID NO:29) located at residues 86-92 of Claspin (SEQ ID NO: 9), as the site mediating binding to Cdh1. Accordingly, in contrast to wild type Claspin, Claspin(ENL) was stable in HeLa cells progressing through G1 (FIG. 2C).

These data show that the Cdh1-binding site in Claspin is not a canonical degron for APC/C substrates (i.e. D-box or KEN box motifs). Non-standard degrons have also been described for Aurora A (the A-box) (Littlepage and Ruderman, 2002) and Orc1 (the O-box) (Araki et al., 2005).

To further illustrate that Claspin is ubiquitylated via APC/C^(Cdh1), the ubiquitylation of Claspin was reconstituted in vitro. The N-terminus of Claspin (amino acids 1-678 of SEQ ID NO:9) was efficiently ubiquitylated only when Cdh1 was present (FIG. 2D and FIGS. 4A-B). FIGS. 4A-B show that the N-terminus of claspin is ubiquitylated in a Cdh1-dependent manner. FIG. 4A shows in vitro ubiquitin ligation assays with ³⁵S-labeled, in vitro transcribed/translated Claspin N-terminus (NT, amino acids 1-678). FIG. 4B shows cyclin B (as a positive control) in vitro ubiquitin ligation assays conducted in the presence or absence of Cdh1 using different UBCs. Samples were incubated at 30° C. and analyzed at the indicated times. The bracket on the right side marks a ladder of bands corresponding to polyubiquitylated proteins. These experiments show that the ubiquitylation of Claspin is dependent on the presence of Cdh1 and is stimulated by Ubc1 (also called E2-25K), which promotes ubiquitylation of APC/C^(Cdh1) substrates by Ubc10. Ubiquitylation of APC/C^(Cdh1) substrates by Ubc10 is described by Rodrigo-Brenni and Morgan, 2007.

In contrast, no Cdh1-dependent ubiquitylation of Claspin(ENL) was observed (FIG. 2D). Similarly, the C-terminus of Claspin (amino acids 679-1333 of SEQ ID NO:9), lacking the Cdh1-binding domain, was not ubiquitylated despite the presence of Cdh1.

Thus, experiments in cell systems including an in vivo model system, and in an in vitro reconstituted model, show that Cdh1 promotes the ubiquitylation and consequent degradation of Claspin in a manner that requires an intact Cdh1-interaction motif.

Example 2

Upon DNA Damage in G2, Usp28 Protects Claspin, but Not Plk1, from APC/C^(Cdh1)-Mediated Degradation

Usp28 deubiquitylates and consequently stabilizes Claspin in response to DNA damage (Zhang et al., 2006). After genotoxic stress, the recognition of Claspin by the SCF^(βTrcp) ubiquitin ligase is impaired due to the inhibition of Plk1 (Mailand et al., 2006; Mamely et al., 2006; Peschiaroli et al., 2006; Smits et al., 2000). Under these conditions, Claspin is continuously ubiquitylated via an unidentified ubiquitin ligase that is different from SCF^(βTrcp). A G2 phase-specific reactivation of APC/C^(Cdh1) after DNA damage has been described in vertebrates (Sudo et al., 2001), but the reason for this reactivation is not known. The present experiments confirm that Cdh1 re-associates with Cdc27 (an APC/C core subunit) in human G2 cells subjected to genotoxic stresses, and that this APC/C^(Cdh1) is active (FIGS. 5A-I).

FIG. 5A shows results after HeLa-S3 cells were synchronized at G1/S using a double thymidine block and then released to allow progression towards G2. At four hours post-release, cells were either left untreated or irradiated (IR) with 10 Gy before harvesting two hours thereafter. Subsequently, whole cell extracts (FIG. 5B, WCE) were immunoprecipitated (IP) with either a mouse anti-Cdh1 antibody or purified, non-specific mouse immunoglobulins (NS IgG), and the indicated proteins were analyzed by immunoblotting. DNA content analyses by FACS (FIGS. 5C, D, and E) were performed to ascertain equal cell cycle distribution of the two samples.

FIG. 5F shows immunoblots after U2OS cells were synchronized at G1/S and then released to allow progression towards G2. At seven hours post-release, cells were pulsed for one hour with either solvent (−) or doxorubicin (DRB) (+) and harvested two hours thereafter. Whole cell extracts (WCE) were immunoprecipitated with a monoclonal anti-Cdc27 antibody or non-specific, purified mouse immunoglobulins (NS IgG). Immunoprecipitates and extracts were immunoblotted with either anti-Cdh1 or anti-Cdc27 antibodies.

FIG. 5G shows results after U2OS cells infected with either an empty virus (EV) or retroviruses expressing FLAG-tagged Cdh1 were synchronized at G1/S and released as in FIG. 5F. At seven hours post-release, cells were pulsed for one hour with either solvent or doxorubicin (DRB) and then harvested at the indicated times. Subsequently, whole cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG resin, and the indicated proteins were analyzed by immunoblotting. DNA content analyses measured by FACS as shown in FIG. 5I and show the cell cycle distribution of the samples.

FIG. 5H shows results after HeLa-S3 cells were synchronized at G1/S using a double thymidine block and then released to allow progression towards G2. At seven hours post release, cells were either left untreated or irradiated (IR) with 10 Gy before harvesting two hours thereafter. Subsequently, cell extracts were immunoprecipitated with an anti-Cdc27 antibody, and immunoprecipitates were used for in vitro ubiquitylation assays using in vitro transcribed/translated Plk1 as a substrate. Note that this ubiquitylation reaction relied only on the presence of Cdh1 co-immunoprecipitated with Cdc27 (i.e. in the absence of in vitro transcribed/translated unlabeled Cdh1, as in all other ubiquitylation assays shown herein) to allow a direct comparison of endogenous APC/C^(Cdh1) under the two different conditions.

In line with these results, experiments were designed to investigate whether APC/C^(Cdh1) targets Claspin during G2 in response to DNA damage, which would explain the need for Usp28. In these experiments, the expression of Cdh1, Usp28, or both Cdh1 and Usp28 were silenced in U2OS cells using previously validated siRNA oligos (SEQ ID NO:21, SEQ ID NO:23). After transfection, cells were synchronized at G1/S and then allowed to progress through the cell cycle. Seven hours after the release from G1/S (when cells were in G2), cells were pulsed with doxorubicin for one hour to induce DNA damage and harvested at different times thereafter (FIGS. 6A-E). The results showed that downregulation of Usp28 resulted in decreased levels of Claspin (FIG. 6A, lanes 14 and 15). However, when both Usp28 and Cdh1 were silenced together, the expression of Claspin was partially restored (FIG. 6A, lanes 19 and 20), indicating that Usp28 counteracts Cdh1-dependent degradation of Claspin.

The immunoblots in FIG. 6A show extracts of U2OS cells that were transfected with the indicated siRNA oligos and synchronized at G1/S using a double thymidine block. Cells were then released from the block to allow progression towards G2. At seven hours post release, cells were pulsed for one hour with either solvent (−) or doxorubicin (DRB) (+) and collected at the indicated times thereafter. Whole cell extracts were analyzed by immunoblotting with antibodies to the indicated proteins. Synchrony was verified by flow cytometry. To facilitate comparison, a gray line separates samples treated with doxorubicin from untreated samples.

The graphs in FIGS. 6B, C, and D show the quantification of the levels of Claspin, Plk1, and Chk1 phosphorylated on Ser317 shown in (FIG. 6A) at the 10 hour timepoint averaged with two additional, independent experiments. The value given for the amount of protein present in the control sample two hours after the end of the doxorubicin pulse was set as 1 (n=3, ±SD).

The immunoblots in FIG. 6E show extracts of U2OS cells that were synchronized and treated with DRB as described in FIG. 6A. Samples were collected at the indicated times and processed for immunoblot analysis with antibodies to the indicated proteins.

To determine if Claspin is protected from proteolysis by Usp28 in DNA-damaged G2 cells, experiments were designed to determine what substrates are targeted for degradation by APC/C^(Cdh1). In these experiments, the levels of 15 G1 substrates of Cdh1 (Claspin, Plk1, Cdc25A, Ubc10, cyclin A, cyclin B, Cdc6, Aurora A, Geminin, Tk1, Tmpk, Skp2, Cks1, Cdc20, and Bub1) were analyzed. U2OS cells were synchronized in G2 and pulsed with doxorubicin, as described for FIG. 6A. Samples were collected at different times thereafter and subjected to immunoblotting. The levels of Plk1 decreased in response to DNA damage (FIG. 6E) in a manner similar to Cdc25A, whose degradation after genotoxic stresses is dependent on SCF^(βTrcp) (Busino et al., 2003; Jin et al., 2003). The other 13 substrates of APC/C^(Cdh1) remained unchanged or showed slight increases in their abundance (FIG. 6E).

Next, it was determined whether Plk1 degradation is dependent on the reactivated APC/C^(Cdh1) complex. These resulted showed that levels of Plk1 were considerably reduced in the presence of doxorubicin at the 10-hour time point compared to the untreated sample (compare lane 3 to lane 5 in FIG. 6A). In contrast, when cells were treated with Cdh1 siRNA oligos (SEQ ID NO:21), Plk1 levels remained unchanged despite the presence of DNA damage (compare lane 3 to lanes 10 and 20 in FIG. 6A). Downregulation of Usp28 did not affect Plk1 levels or Plk1 half-life (FIG. 6A and FIG. 7A), indicating that Usp28 does not oppose ubiquitylation of Plk1 as it does with Claspin.

FIGS. 7A-C show Usp28 downregulation destabilizes Claspin but not Plk1. U2OS cells were transfected with the indicated siRNA oligos and synchronized at G1/S using a double thymidine block. Cells were then released from the block to allow progression towards G2. Seven hours post release, doxorubicin (DRB) (+) and cycloheximide (CHX) were added to the cells, which were collected at the indicated times thereafter. Cell lysates were then immunoblotted with antibodies to the indicated proteins. FIG. 7B-C show the quantification of the levels of Plk1 and Claspin shown in FIG. 7A.

FIG. 7D shows downregulation of either Cdh1 or Cdc14B results in the stabilization of Plk1 and in the destabilization of Claspin in DNA damaged cells. U2OS cells were transfected with the indicated siRNA oligos and synchronized at G1/S using a double thymidine block. Cells were then released from the block to allow progression towards G2. Seven hours post-release, cells were pulsed for one hour with either solvent (−) or doxorubicin (DRB) (+). Sixteen hours later, cycloheximide (CHX) was added to the culture for the indicated times prior to harvesting. Cell lysates were then immunoblotted with antibodies to the indicated proteins. FIG. 7E-F show the quantification of the levels of Plk1 and Claspin shown in top panels. FIG. 7G shows Cdc14B mRNA levels of cells used in top panels (FIGS. 7D-E) were analyzed at the “0 hrs” time point using real time PCR in triplicate measurements (±SD). The value given for the amount of Cdc14B mRNA present in cells treated with control oligos was set as 1.

FIG. 7H shows Expression of Cdh1(4xA) does not allow Plk1 stabilization after DNA damage, despite the downregulation of Cdc14B. U2OS cells were retrovirally infected with either wild type Cdh1 or Cdh1(4xA) mutant and subsequently transfected with siRNA oligos directed against Cdc14B mRNA. Cells were synchronized at G1/S using a double thymidine block and then released from the block to allow progression towards G2. Seven hours post release, cells were pulsed for one hour with either solvent (−) or doxorubicin (DRB) (+). Sixteen hours later, cycloheximide (CHX) was added to the culture for the indicated times prior to harvesting. Cell lysates were then immunoblotted with antibodies to the indicated proteins.

FIG. 7I-J show the quantification of the levels of Plk1 and Claspin shown in top panels (FIG. 7H). FIG. 7K shows Cdc14B mRNA levels of cells used in top panels analyzed at the “0 hrs” time point using real time PCR in triplicate measurements (±SD). The value given for the amount of Cdc14B mRNA present in the sample expressing wild type Cdh1 and treated with control oligos was set as 1.

Knockdown of Cdh1 and/or Usp28 had no effect on Cdc25A (FIG. 6A). Interestingly compared to samples treated with control oligos, downregulation of Cdh1 induced a reduction in the levels of Claspin and in the activating phosphorylation of Chk1 on Ser317 (FIGS. 6A, B). This last result shows that APC/C^(Cdh1) activity is necessary to sustain Claspin expression in response to genotoxic stress in G2. Since Plk1 promotes Claspin degradation, the stabilization of Plk1, which occurred in cells where Cdh1 expression was silenced, may explain why Claspin levels decrease under these conditions.

The above results show that APC/C^(Cdh1) targets Claspin and Plk1 in response to DNA damage in G2 and that Usp28 selectively counteracts this action on Claspin. In addition, these results suggest that the APC/C^(Cdh1)-mediated degradation of Plk1 promotes Claspin stabilization and the consequent activation of Chk1.

Example 3

Cdh1-Dependent Degradation of Plk1 is Required for an Efficient DNA Damage-Induced G2 Checkpoint

A destruction box in Plk1 is required for the APC/C^(Cdh1)-dependent degradation of Plk1 during late M and in G1 (Lindon and Pines, 2004). A stable Plk1 mutant [Plk1(R337A,L340A)] was generated to investigate the biological function of Plk1 degradation in response to DNA damage in G2. U2OS cells were retrovirally infected with either wild type Plk1 or Plk1(Plk1 R337A, L340A) (i.e., the mutant Plk1) were synchronized in G2, pulsed for one hour with doxorubicin, and followed for an additional three hour period. These results showed that Plk1(R337A,L340A) was not degraded following treatment with doxorubicin, whereas a significant decrease in the levels of wild type Plk1 was observed (FIGS. 8A, B, C, and D). The difference in Plk1 levels corresponded to the difference in the half-lives of wild type Plk1 versus Plk1(R337A,L340A) (FIG. 8E, F). This finding shows that DNA damage-induced degradation of Plk1 in G2 is mediated by its Cdh1-binding site.

U2OS cells retrovirally infected with the indicated Plk1 constructs were synchronized and treated with DRB as described in FIG. 6A. Cells were then collected at the indicated times and immunoblotted with antibodies to the indicated proteins. To facilitate comparison, a gray line separates samples treated with doxorubicin from untreated samples. The immunoblots are shown in FIG. 8A.

The graphs in FIG. 8B-D show the quantification of the levels of Claspin, Plk1, and Chk1 phosphorylated on Ser317 shown in FIG. 8A at the 10 hour timepoint averaged with an additional, independent experiment. The value given for the amount of protein present in the control sample two hours after the end of the doxorubicin pulse was set as 1 (n=2).

U2OS cells were infected, synchronized, and pulsed with doxorubicin as in FIG. 8A, except that cycloheximide (CHX) was added seven hours after release from G1/S. At different times after the addition of CHX, cells were collected, and cell lysates were immunoblotted with antibodies to the indicated proteins. The immunoblots are shown in FIG. 8E.

The graph in FIG. 8F shows the quantification of the levels of wild type Plk1 and Plk1(R337A, L340A) shown in FIG. 8E averaged with two additional, independent experiments (n=3, ±SD).

U2OS cells infected with either an empty virus (EV) or retroviruses encoding wild type Plk1, Plk1(R337A, L340A), or Cdc25A(S82A) were synchronized and treated with DRB as described in FIG. 8A. Thereafter, cells were incubated in fresh medium containing nocodazole to trap cells in mitosis. Samples were then collected at the indicated times, and the percentage of mitotic cells was monitored by immunodetection of Histone H3 phosphorylated on Ser10 using flow cytometry (n=3, ±SD). The graph of the flow cytometric results is shown in FIG. 8G.

U2OS cells treated as in (FIG. 8A) were collected 23 hours after release from G1/S (16 hours after the doxorubicin pulse), and then cell lysates were immunoblotted with antibodies to the indicated proteins. The immunoblots are shown in FIG. 8H.

Following DNA damage, the lack of Plk1 (R337A, L340A) degradation was associated with decreased levels of Claspin and reduced phosphorylation of Chk1 (FIGS. 3A, B), similar to what is observed when Cdh1 is silenced (FIGS. 6A, B). Since Plk1 initiates the SCF^(βTrcp)-dependent degradation of Claspin following recovery from DNA damage stresses, these data argue for a role of Cdh1 in limiting Plk1 levels to preserve Claspin stability and Chk1 activation in response to DNA damage in G2.

To investigate the impact of stable Plk1 on the maintenance of the checkpoint, U2OS cells treated as in FIG. 8A were incubated with nocodazole (to trap cells in mitosis) and followed for longer time points (up to 31 hours after release from G1/S, i.e. 24 hours after the pulse with doxorubicin) (FIG. 8G). In addition to cells expressing wild type Plk1 or Plk1(R337A,L340A), this experiment included cells expressing a stable Cdc25A(S82A) mutant, which is known to impair the checkpoint response to DNA damage (Busino et al., 2003). Mitotic entry was determined by immunostaining for Ser10 phosphorylated Histone H3. Cells infected with an empty viral vector showed only marginal mitotic entry, indicative of an intact G2 checkpoint, while cells expressing wild type Plk1 or Cdc25A (S82A) displayed an increase in cells entering mitosis. Strikingly and surprisingly, mitotic entry was significantly more pronounced in cells expressing Plk1(R337A, L340A), indicating that Cdh1-mediated degradation of Plk1 is a critical step to establish and maintain the DNA damage checkpoint in G2. The relatively moderate elevation of mitotic entry observed when wild type Plk1 was expressed is likely due to the increased demand on the proteolytic machinery to degrade the excess of Plk1.

Importantly, the expression of a stable Claspin mutant (lacking the βTrCP degron) prevented Plk1(R337A, L340A) from bypassing the checkpoint (FIG. 9), confirming that the induced degradation of Claspin is a major mechanism for Plk1 to induce premature mitosis. In FIG. 9, the experiment was performed as in FIG. 8G, except U2OS cells were also infected with retroviral constructs encoding both Plk1(R337A,L340A) and either a Claspin mutant lacking the βTrcp degron [Claspin(S30/34A)] (Peschiaroli et al., 2006) or a Wee1 mutant lacking the βTrcp degron [Wee1(S53/123A)] (Watanabe et al., 2004). The percentage of mitotic cells was monitored by immunodetection of Histone H3 phosphorylated on Ser10 using flow cytometry (n=3, ±SD for the first three bars and n=2 for the last one).

To elucidate the molecular mechanisms by which cells expressing Plk1 (R337A, L340A) are able to bypass this checkpoint, cell extracts were also analyzed by immunoblotting (FIG. 8H). All major events downstream from Plk1, which ultimately lead to the activation of Cdk1 and entry into mitosis, were observed in DNA-damaged cells expressing the stable Plk1 mutant but not wild type Plk1. FIG. 8H shows that compared to wild type Plk1 (lane 2), Plk1(R337A, L340A) expression (lane 4) resulted in: (i) degradation of Claspin, with the consequent reduction in activating phosphorylation of Chk1; (ii) degradation of the Cdk1 inhibitor Wee1 [another SCF^(βTrcp) substrate, whose degradation is promoted by Plk1 (Watanabe et al., 2004)]; (iii) elevated Cdc25A levels (likely due to low Chk1 activity); (iv) dephosphorylation of Cdk1 on Tyr15 (likely due to low levels of Wee1 and high Cdc25A expression); (v) lower p21 levels; and (vi) phosphorylation of Histone H3 on Ser10. Thus, cells entered mitosis despite the presence of DNA damage, visualized by the presence of H2AX phosphorylation (bottom panel in FIG. 8H and FIG. 10A-F).

FIGS. 10A-F show U2OS cells infected with a retrovirus encoding Plk1(R337A,L340A) were synchronized at G1/S using a double thymidine block. Cells were then released from the block to allow progression towards G2. At seven hours post release, cells were pulsed for one hour with either solvent (−) or doxorubicin (DRB) (+). Thereafter, cells were incubated in fresh medium containing nocodazole to trap cells in mitosis. Samples were collected 23 hours after release from G1/S and analyzed by indirect immunofluorescence with an antibody specific for phosphorylated H2AX (FIGS. 10A-B) and DAPI (to visualize condensed DNA) (FIG. 10C-D). Chromosomes of all cells that entered mitosis after DRB treatment were positive for phospho-H2AX, showing that despite unrepaired DNA lesions cells expressing stable Plk1 reached mitosis. The merged results overlaying both stains are shown in FIGS. 10E-F.

Therefore, these data show that Cdh1-driven degradation of Plk1 in response to DNA damage is vital to maintain an efficient G2 checkpoint.

Example 4

Cdc14B relocates to the nucleus and associates with Cdh1 in G2 in a DNA-damage-dependent manner.

To be able to associate with and activate APC/C in late mitosis, Cdh1 must be dephosphorylated at sites that were phosphorylated by CDKs during S and G2 (Lukas et al., 1999; Mitra et al., 2006; Sorensen et al., 2001). The present data indicates that APC/C^(Cdh1) is reactivated in G2 following DNA damage to target Plk1 and further indicates that a phosphatase may be required to remove the inhibitory phosphates from Cdh1. Studies in yeast have shown that Cdc14 dephosphorylates Cdh1 in late mitosis (D'Amours and Amon, 2004; Sullivan and Morgan, 2007). Two Cdc14 paralogs exist in mammals: Cdc14A and Cdc14B. Whereas Cdc14A is localized to the centrosomes, Cdc14B localizes to the nucleolus (Cho et al., 2005; Mailand et al., 2002). As a first step to study whether one of the Cdc14 phosphatases may dephosphorylate and activate Cdh1 in G2 phase in response to a genotoxic stress, experiments were designed to illustrate their subcellular localizations after treatment with doxorubicin or ionizing radiation. These experiments showed that Cdc14B was initially localized to the nucleolus; however surprisingly, following DNA damage, it moved to the nucleoplasm (FIG. 11A). In contrast, no change in subcellular localization was observed for Cdc14A. FIG. 11A shows that Cdc14B moves from the nucleolus to the nucleoplasm in response to DNA damage. U2OS cells transfected with a construct expressing GFP-tagged Cdc14B were synchronized as described in FIG. 6A, and then either treated with DRB [+/− caffeine (CAF)] or subjected to ionizing radiations (IR). Cells were then collected and analyzed by direct immunofluorescence. FIG. 11A shows micrographs of representative cells showing the subcellular localization of Cdc14B. FIG. 11B shows the quantification of cells with nuclear Cdc14B fluorescence at the indicated times post pulse with doxorubicin (n=3, ±SD).

FIG. 11C shows that Cdc14B translocation to the nucleus is independent of ATM. The experiment was performed as in FIG. 11A, except that asynchronous ATM+/+ and ATM−/− fibroblasts were used (n=2). The quantification of the cells with nuclear fluorescence is shown in FIG. 11D.

FIG. 11E shows the graphic results of an experiment performed as in FIG. 11A, except that GFP-tagged Cdc14B(T426A) was used (n=3).

FIG. 11F shows Cdh1 binds to Cdc 14B in a DNA damage-dependent manner. U2OS cells infected with either an empty retrovirus (EV) or viruses encoding FLAG-tagged Cdc14B were synchronized and treated with DRB as described in FIG. 11A. Cells were collected two hours later, and whole cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG resin. The indicated proteins were detected by immunoblotting.

Interestingly, the nuclear localization of Cdc14B did not depend on ATM, as relocalization from the nucleolus was also observed in the presence of caffeine (FIG. 11A) and in ATM−/− fibroblasts (FIG. 11C). Human Cdc14B (SEQ ID NO:15) has three ATM phosphorylation consensus sites (one SQ and two TQs), two of which are not conserved in many other mammals. In contrast, the third site (Thr426) is conserved in all vertebrates. The Thr426 was mutated to Ala and this mutant Cdc14B(T426A) continued to move to the nucleus after DNA damage (FIG. 11E), confirming that ATM does not play a role in Cdc14B translocation.

Next, to test whether Cdc14B and Cdh1 physically interact in response to DNA damage in G2, co-immunoprecipitation experiments in G2 U2OS cells were performed. Surprisingly, an interaction between the two proteins was only observed after treatment with doxorubicin (FIG. 11F). Thus, during the G2 DNA damage response, Cdc14B moves to nucleus and binds Cdh1.

Example 5 Cdc14B Activates APC/C^(Cdh1) After DNA Damage in G2

The finding that Cdc14B and Cdh1 interact in a DNA damage-dependent manner in G2 indicated that Cdc14B is involved in the reactivation of APC/C^(Cdh1). To further test this hypothesis, the effect of silencing Cdc14B was determined. U2OS cells were transfected with previously validated siRNA oligos directed against either Cdc14B (SEQ ID NO:22) or Cdh1 (SEQ ID NO:21) and then synchronized. Upon reaching G2 (seven hours after release from G1/S), cells were pulsed with doxorubicin and collected at different times points thereafter (FIG. 12A).

In FIG. 12A U2OS cells were transfected with the indicated siRNA oligos and the cells were synchronized and treated with DRB as described in FIG. 6A. The cells were collected at the indicated times, and cell lysates were analyzed by immunoblotting with antibodies to the indicated proteins.

The graphs in FIGS. 12B, C, and D show the quantification of Claspin, Plk1, and Chk1 phosphorylated on Ser317 shown in FIG. 5A, at the 10 hour timepoint averaged with an additional, independent experiment. The value given for the amount of protein present in the control sample two hours after the end of the doxorubicin pulse was set as 1 (n=2).

U2OS cells, transfected with the indicated siRNA oligos, were treated as in FIG. 12A. Twenty-three hours after release from G1/S (16 hours after the doxorubicin pulse), cells were collected, and cell lysates were analyzed by immunoblotting with antibodies to the indicated proteins. The immunoblots are shown in FIG. 12E.

FIG. 12F shows Cdc14B mRNA levels of cells used in FIG. 5A and FIG. 5C that were analyzed eight hours after release from G1/S using real time PCR in triplicate measurements (±SD). The value given for the amount of Cdc14B mRNA present in the sample treated with control oligos was set as 1.

Strikingly, silencing of Cdc14B resulted in the stabilization of Plk1 to an extent comparable to that obtained from silencing Cdh1 (FIG. 6A and FIGS. 12A-B). Moreover, reduced levels of Claspin and impaired phosphorylation of Chk1 at Ser317 were observed in the Cdc14B knockdown samples, suggesting that the elevated Plk1 levels produced an effect on downstream targets. Thus, the presence of Cdc14B appears vital for Plk1 degradation in G2 upon genotoxic stress.

To investigate the impact of silencing Cdc14B on the maintenance of the G2 checkpoint, U2OS cells treated as in FIG. 12A were followed for a longer time course (up to 23 hours after release from G1/S). Even at these late time points, Plk1 was stabilized when Cdc14B was downregulated (FIG. 12E and FIG. 7D). Importantly, similar to what was observed when the stable Plk1 mutant was expressed (FIG. 8H), events downstream of Plk1, which lead to entry into mitosis, were observed in the Cdc14B knockdown samples (FIGS. 12E, F): increased degradation of Claspin and Wee1, reduction in the activating phosphorylation of Chk1, elevated Cdc25A levels, reduction in the inactivating phosphorylation of Cdk1, and phosphorylation of Histone H3 on Ser10.

Thus, Cdc14B, similar to Cdh1, is critical to establish and maintain the DNA damage checkpoint in G2. Interestingly, while downregulation of Cdc14B induced checkpoint bypass, the combination of Cdh1 and Cdc14B siRNAs had no additional effect (FIG. 14), showing that both proteins function in the same pathway.

The phosphatase activity of Cdc14B towards Cdh1 is predicted to occur on sites that are phosphorylated by CDKs. The four sites phosphorylated in Cdh1 by CDKs during S and G2 have been previously described (Lukas et al., 1999; Sorensen et al., 2001). To further investigate the role of Cdc14B in activating Cdh1 in G2 upon DNA damage, U2OS cells were infected with retroviruses expressing either wild type Cdh1 or Cdh1(4xA), a constitutively active, phosphorylation-deficient Cdh1 mutant, in which the four CDK phosphorylation sites are mutated to Alanine. Subsequently, these cells were subjected to experiments analogous to those shown in FIG. 12-A-F.

FIG. 13A shows results after U2OS cells were retrovirally infected with either wild type Cdh1 or Cdh1(4xA) mutant and subsequently transfected with the indicated siRNA oligos. Cells were then synchronized and treated with DRB as described herein for FIG. 6A, collected at the indicated times, and immunoblotted with antibodies to the indicated proteins. To facilitate comparison, a gray line separates samples treated with doxorubicin from untreated samples.

The graphs in FIGS. 13B, C, and D show the quantification of the levels of Claspin, Plk1, and Chk1 phosphorylated on Ser317 shown in (FIG. 13A) at the indicated timepoint averaged with an additional, independent experiment. The value given for the amount of protein present in the control sample two hours after the end of the doxorubicin pulse was set as 1 (n=2).

The immunoblots in FIG. 13E show U2OS cells expressing wild type Cdh1 or Cdh1(4xA) that were transfected with the indicated siRNA oligos and treated as in (FIG. 13A). Twenty-three hours after release from G1/S (16 hours after the doxorubicin pulse), cells were collected, and cell lysates were analyzed by immunoblotting with antibodies to the indicated proteins.

In FIG. 14, the experiment was performed as in FIG. 13E, except that U2OS cells were also transfected with siRNA oligos targeting both Cdh1 and Cdc14B. The percentage of mitotic cells was monitored by immunodetection of Histone H3 phosphorylated on Ser10 using flow cytometry (n=3). These results show that co-silencing of Cdh1 and Cdc14B is not synergistic in bypassing the checkpoint.

In FIG. 13F Cdc14B mRNA levels of cells used in (FIG. 13A) and (FIG. 13E) were analyzed eight hours after release from G1/S using real time PCR in triplicate measurements (±SD). The value given for the amount of Cdc14B mRNA present in the sample expressing wild type Cdh1 and treated with control oligos was set as 1.

In FIG. 13G synchrony in G2 was ascertained by flow cytometry for cells used in (FIG. 13A) at the time of doxorubicin treatment. Identical synchrony in G2 was obtained for cells used in (FIG. 13E).

In FIG. 13H, the immunoblotting experiment was performed as in (FIG. 13A), except that an in vivo labeling with ³²P-orthophosphate was performed during the last three hours before cells were collected. Cdh1 was then immunoprecipitated under denaturing conditions, resolved by SDS-PAGE, and visualized by autoradiography (upper panel) or immunoblotting (bottom panel). FIG. 13I shows Cdc14B mRNA levels analyzed by real time PCR.

As in non-infected cells, the silencing of Cdc14B in cells expressing wild type Cdh1 resulted in the stabilization of Plk1 in response to DNA damage and in downstream events regulated by Plk1 (FIGS. 13A-G). In marked contrast, silencing of Cdc14B did not stabilize Plk1 in cells expressing Cdh1(4xA) (FIGS. 13A-G and FIG. 7H). Consequently, Claspin was not degraded, and Chk1 was efficiently phosphorylated upon DNA damage. Moreover, despite the downregulation of Cdc14B, cells expressing Cdh1(4xA) displayed high levels of Wee1, inactivating phosphorylation of Cdk1, high levels of p21, and low levels of phosphorylation of Histone H3 on Ser10 (FIG. 13E, compare lane 2 and 4), showing that in the presence of a constitutively active Cdh1 mutant, Cdc14B becomes dispensable for sustaining the G2 checkpoint.

Importantly, these results show that after DNA damage, the in vivo phosphorylation of wild type Cdh1—but not that of Cdh1(4xA)—increased after Cdc14B silencing (FIG. 13H), indicating that in response to genotoxic stress, Cdc14B dephosphorylates Cdh1 on the four sites phosphorylated by Cdk2.

These results indicate that upon DNA damage in G2, Cdh1 is the relevant target of Cdc14B and that CDK-mediated phosphorylation of Cdh1 is the relevant constraint that is removed by Cdc14B.

Example 6

The Cdc14B-Cdh1-Plk1 Axis is Deregulated in Human Tumors

These data show that the Cdc14B-Cdh1-Plk1 axis controls the G2 DNA damage checkpoint (see model in FIG. 15A). FIG. 15A is a schematic model of the G2 DNA damage response checkpoint. Black signifies activated forms of the respective proteins, and gray indicates inactive forms or degraded proteins. After induction of double strand breaks (DSB) in G2, ATM activates Chk2. In addition, ATM activates certain exonucleases to induce DSB resection, resulting in RPA coated, single-stranded DNA (ssDNA), which contributes to the recruitment of ATR. In parallel, Cdc14B is released from the nucleolus to the nucleoplasm, activating APC/C^(Cdh1), which in turn targets Plk1 for proteasomal degradation. Because of the low levels of Plk1, phosphorylation of Claspin and Wee1 is reduced, preventing βTrcp-mediated degradation. Claspin is protected from APC/C^(Cdh1)-mediated degradation by Usp28. Stable Claspin promotes the ATR-mediated activation of Chk1, which, together with Chk2, targets Cdc25A (inducing its degradation) and p53 (promoting its stabilization and consequent induction of p21). As a result, Cdk1 activity is attenuated, and cells arrest in G2. Stable Wee1 contributes to this inhibition by directly phosphorylating Cdk1. The reduction in Cdk1 activity further removes the constraints on APC/C^(Cdh1) activity.

FIGS. 15B-D show poor patient survival correlates with low levels of Cdc14B and Cdh1 and high levels of Plk1 (gray). Data evaluated from the Rembrandt glioma database from the worldwide web (NCI-NIH) generated Kaplan-Meier survival curves of 219 patients with gliomas of all histological grades grouped by gene expression levels of Cdc14B (top plot), Cdh1 (middle plot), and Plk1 (bottom plot). The associated P values are shown for each plot.

To investigate a potential involvement of this pathway in human cancer, the Oncomine database was searches for the differential expression of Cdc14B, Cdh1, and Plk1 in normal versus tumor tissues. Analysis of the database results show the expression of Cdc14B (always) and Cdh1 (in most cases) to be significantly decreased in many common tumors, such as breast, prostate, ovary, liver, and brain tumors (FIGS. S10A-L). FIGS. 16A-R show the analysis of mRNA levels of Cdc14B, Cdh1, and Plk1 in different human tumors. All raw data was provided by the Oncomine database. The associated P values are shown for each study.

FIGS. 16A, G, and M show initial data from (Chen et al., 2002) reanalyzed to show expression levels of Cdc14B (A), Cdh1 (G), and Plk1 (M) in non-tumor liver (Normal) and hepatocellular carcinoma (Carcinoma).

FIGS. 16B, H, and N show initial data from (Richardson et al., 2006) reanalyzed to show expression levels of Cdc14B (B), Cdh1 (H), and Plk1 (N) in normal breast (Normal) and breast carcinoma (Carcinoma).

FIGS. 16C, I, and O show initial data from (Sotiriou et al., 2006) reanalyzed to show expression levels of Cdc14B (C), Cdh1 (I), and Plk1 (Q) in Grade 1, Grade 2, and Grade 3 breast cancers.

FIGS. 16D, J, and P show initial data from (Tomlins et al., 2007) reanalyzed to show expression levels of Cdc14B (D), Cdh1 (J), and Plk1 (P) in benign prostate (Normal) and prostate carcinoma (Carcinoma).

FIGS. 16E, K, and Q show initial data from (Hendrix et al., 2006) reanalyzed to show expression levels of Cdc14B (E), Cdh1 (K), and Plk1 (Q) in normal ovary (NO), ovarian endometrioid adenocarcinoma (OEA), ovarian serous adenocarcinoma (OSA), and ovarian mucinous adenocarcinoma (OMA).

FIGS. 16F, L, and R show initial data from (Sun et al., 2006) reanalyzed to show expression levels of Cdc14B (F), Cdh1 (L), and Plk1 (R) in normal brain (NB), glioblastoma multiform (GBM), astrocytoma (AS), and oligodendroglioma (OD).

Conversely, levels of Plk1 were consistently and significantly increased in most tumors (FIGS. 16M-R). Importantly, the decrease in Cdc14B expression and the increase in Plk1 levels significantly correlate with the grade of breast cancer.

Finally, downregulation of Cdc14B and Cdh1 and/or upregulation of Plk1 correlate with survival in glioma patients (FIG. 15B). These results suggest that a failure in the Cdc14B-Cdh1-Plk1 axis is likely to impair the G2 DNA damage checkpoint, with the consequent acquisition of genomic instability.

Discussion of Results

Following genotoxic stresses, Claspin promotes ATR-mediated phosphorylation and activation of Chk1 (Harper and Elledge, 2007). In G2, during the recovery from DNA replication stress or DNA damage, Claspin is phosphorylated by Plk1 and consequently degraded via SCF^(βTrcp) (Mailand et al., 2006; Mamely et al., 2006; Peschiaroli et al., 2006). This process contributes to turning off the checkpoint to promote the entry of cells into mitosis. SCF^(βTrcp)-dependent ubiquitylation of Claspin remains active at least until prometaphase; however, Claspin reaccumulates only at the G1/S transition of the next cell cycle, suggesting that Claspin degradation is active during G1.

Several substrates degraded via SCF ligases in specific phases of the cell cycle are eliminated by the means of APC/C at different times. For example, p21 and M11 are degraded at G1/S via SCF^(Skp2) and at G2/M via APC/C^(Cdc20) (Amador et al., 2007; Liu et al., 2007). Similarly, Cdc25A, whose degradation is mediated by SCF^(βTrcp) during S, is eliminated in G1 via APC/C^(Cdh1) (Busino et al., 2003; Donzelli et al., 2002; Jin et al., 2003). The present results illustrate that, in contrast to degradation of Claspin in G2 via SCF^(βTrcp), Claspin proteolysis in G1 cells is directed by APC/C^(Cdh1) (FIGS. 1-4).

APC/C^(Cdh1) maintains the G0/G1 state by ensuring that many positive regulators of S and M do not accumulate prematurely, a function important for genome stability (Guardavaccaro and Pagano, 2006; Peters, 2006). Claspin plays a poorly understood, positive role in DNA replication, which appears distinct from its role in checkpoint signaling. In fact, not only does expression of Claspin represent a reliable marker of cell proliferation in both human cancer and normal tissues (Tsimaratou et al., 2007), but Claspin overexpression has also been shown to increase cell proliferation (Lin et al., 2004). The present results support that Claspin represents another substrate that APC/C^(Cdh1) keeps at low levels during G1 to avoid premature entry into S.

This initial result proved to be an invaluable portal to gain entry into the signaling network that mediates the G2 checkpoint, starting from the following observation. Upon DNA damage in G2, Claspin is no longer phosphorylated on its degron, blocking recognition by βTrcp (Peschiaroli et al., 2006). Therefore, the present experiments were undertaken to determine why Usp28 is required to deubiquitylate and stabilize Claspin upon DNA damage (Zhang et al., 2006). The present results demonstrate that APC/C^(Cdh1) is reactivated in response to DNA damage (FIG. 5), thereby inducing ubiquitylation of Claspin, which is neutralized by Usp28 (FIGS. 6A-B).

These results lead to a novel question. If Claspin is ubiquitylated in response to DNA damage as a byproduct of APC/C^(Cdh1) activation, but is protected from degradation by Usp28, what are the relevant substrate(s) that are degraded via APC/C^(Cdh1) under these conditions? To address this question, in the present experiments, 15 substrates that are known to be targeted by this ligase in G0/G1 were surveyed and the data show that only two were downregulated: Plk1 and Cdc25A (FIG. 6E). The latter is known to be degraded via SCF^(βTrcp) after genotoxic stresses (Busino et al., 2003; Jin et al., 2003), and, in fact, its levels did not promptly increase when Cdh1 was silenced (FIG. 6A). In contrast, Plk1 degradation was abolished when Cdh1 levels were lowered by siRNA (FIGS. 6A, B, C, D, and FIGS. 12A-E), showing that Cdh1 promotes Plk1 degradation. The lack of Claspin phosphorylation and consequent stabilization (Peschiaroli et al., 2006) is attributed to the inactivation of Plk1 that occurs after DNA damage (Smits et al., 2000), but how Plk1 is inactivated had remained largely unknown. The present experiments show that Plk1 is degraded via APC/C^(Cdh1) following DNA damage. Downregulation of Cdh1 or the expression of a stable Plk1 mutant both resulted in the downregulation of Claspin in G2 DNA-damaged cells (FIGS. 6A, B, FIGS. 8A, H and FIGS. 5A, E). Thus, APC/C^(Cdh1) exerts a dual control on Claspin stability: during G1, it promotes Claspin degradation, whereas after genotoxic stresses in G2, it allows Claspin stabilization by mediating the degradation of Plk1, which in turn disables Claspin proteolysis via SCF^(βTrcp).

Knockdown of Cdh1 or the expression of a Cdh1-insensitive mutant of Plk1 also resulted in additional defects in the following cellular responses to DNA damage: activating phosphorylation of Chk1 (that correlates with the accumulation of Claspin), Cdc25A degradation, accumulation of Wee1, inactivating phosphorylation of Cdk1, induction of p21, and low levels of phosphorylation of Histone H3 on Ser10 (FIG. 6A, FIGS. 8A, H, FIGS. 12A, E, and FIG. 15A). Importantly, the present results show that APC/C^(Cdh1)-mediated degradation of Plk1 is essential for the establishment of an efficient G2 checkpoint that prevents entry into M upon genotoxic stresses (FIGS. 8G, H).

The degradation of Cdc25A and induction of p21 represent two established mechanisms that contribute to the inactivation of Cdk1 in response to DNA damage. The present results show for the first time that APC/C^(Cdh1)-mediated degradation of Plk1 is upstream of these events (see model in FIG. 15A). Moreover, by promoting Wee1 stabilization, the degradation of Plk1 also induces a direct inhibition of Cdk1. Thus, Plk1 proteolysis via APC/C^(Cdh1) appears to be necessary for effective inhibition of Cdk1 after genotoxic stresses.

The molecular mechanisms leading to APC/C^(Cdh1) reactivation in response to DNA damage were also investigated. CDK-dependent phosphorylation of Cdh1 dissociates Cdh1 from APC/C to prevent its activation from G1/S until anaphase. In yeast, Cdh1 activation in anaphase involves Cdh1 dephosphorylation by the Cdc14 phosphatase, but whether this mechanism is conserved in mammals is unknown. Interestingly, the levels of Cdc14 in yeast and frog do not oscillate during the cell cycle, but the protein is sequestered in the nucleolus until mitosis (D'Amours and Amon, 2004; Kaiser et al., 2004). Likewise, the expression of human Cdc14B mRNA does not change during cell cycle progression (Whitfield et al., 2002), and Cdc14B protein is localized to the nucleolus (Cho et al., 2005). The present results show that in response to DNA damage, human Cdc14B translocates from the nucleolus to the nucleoplasm, where it physically binds and activates Cdh1 (FIGS. 11-13). This result explains why Cdc14B is not expressed exclusively at G2/M, like other mitotic regulators. Rather, it is sequestered in the nucleolus during interphase, allowing prompt release in response to genotoxic stresses.

Since the expression of Cdh1(4xA) makes Cdc14B dispensable upon DNA damage in G2, and silencing of Cdc14B increases the phosphorylation of wild type Cdh1 but not that of Cdh1(4xA) (FIG. 13), it is proposed that Cdc14B activates Cdh1 by reversing the CDK-mediated phosphorylation of Cdh1.

Interestingly, Cdc14B release from the nucleolus is not dependent on ATM (whose activation is considered the most upstream signaling event after chromosomal breakage) (FIGS. 11A-E). The mechanism of mitotic release/activation of Cdc14 is relatively well understood in yeast, where the process is initiated by the “early anaphase release network” and completed by the “mitotic exit network” (D'Amours and Amon, 2004; Sullivan and Morgan, 2007).

In addition to Claspin, other Cdh1 substrates (e.g., cyclin A, cyclin B, and Geminin) are stable in damaged cells despite the reactivation of APC/C^(Cdh1). Nevertheless, they are not destabilized when either Cdh1 and/or Usp28 are downregulated (FIG. 6A). Thus, these results indicate that two pools of APC/C^(Cdh1) exist in G2. One pool (targeting cyclins and Geminin) is inactive both in the presence and absence of genotoxic stress, likely because it is inhibited by Emi1. (In fact, FIG. 5F shows that Emi1 binding to Cdc27 is not influenced by DNA damage.) The other pool (targeting Claspin and Plk1) is inhibited by phosphorylation and is reactivated in response to DNA damage, with Claspin ubiquitylation counteracted by Usp28.

While DUBs reverting protein modification by ubiquitin (e.g., monoubiquitylation and polyubiquitylation via Lys63) and ubiquitin-like molecules that do not target proteins for degradation are simple to envision, it is more difficult to envision why proteins should be stabilized by deubiquitylation rather than by lack of ubiquitylation. The present results show that in some cases, regulation by DUBs is necessary to counteract the activity of ubiquitin ligases on selective substrates. The specificity of DUBs, however, is poorly understood, and it is currently unknown how Usp28 targets Claspin but not Plk1.

The present analysis of the available databases also shows that the levels of Cdc14B, Plk1, and Cdh1 are deregulated in human tumors, and this deregulation correlates with the survival of cancer patients (FIG. 15B and FIG. 16). Abnormal levels or activity of these proteins is likely to disrupt orderly cell cycle progression. Furthermore, the present results illustrate that a failure in the Cdc14B-Cdh1-Plk1 axis induces an inefficient G2 DNA damage checkpoint, with the consequent risk of genomic instability.

In conclusion, while it was previously appreciated that the major function of APC/C^(Cdh1) is the maintenance of the G0/G1 state, the present results demonstrate that in response to DNA damage, APC/C^(Cdh1) is reactivated to allow the elimination of the promitotic kinase Plk1. Importantly, APC/C^(Cdh1)-mediated degradation of Plk1 is essential for the establishment and maintenance of an efficient G2 checkpoint. Finally, the present data provide evidence that APC/C^(Cdh1) is activated by Cdc14B released from the nucleolus upon genotoxic stresses. These findings indicate that the Cdc14B-Cdh1-Plk axis is a hub in the G2 DNA damage response in mammalian cells that is important for the inhibition of Cdk1 and the prevention of entry into mitosis. In contrast to the βTrCP-mediated degradation of Cdc25A and the p53-dependent induction of p21, Cdc14B, Plk1, and Cdh1 are well conserved from yeast to human, suggesting that the regulatory network described herein represents an “ancient” response to genotoxic stress that is conserved along evolution.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

While the compositions and methods of this invention have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention as defined by the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

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1. A method of sensitizing a cancer cell to apoptosis or cell death comprising contacting the cell with an effective amount of siRNA which has the sequence consisting of SEQ ID NO:
 22. 2. A method of sensitizing a cancer cell to apoptosis or cell death comprising contacting the cell with an effective amount of an inhibitor of Cdc14B and an inhibitor of Cdh1.
 3. The method of claim 2, wherein the inhibitor of Cdc14B is an siRNA.
 4. The method of claim 2, wherein the inhibitor of Cdh1 is an siRNA.
 5. The method of claim 4, wherein the siRNA has the sequence consisting of SEQ ID NO:
 21. 6. The method of claim 2, wherein the amount of Plk1 protein in the cell is increased compared to the amount of Plk1 protein in the cell prior to use of the inhibitor. 